Drug-immobilized particles and a process of purifying proteins

ABSTRACT

The invention includes a microsphere comprising styrene-glycidyl methacrylate polymer, and methods for isolation, purification and identification of receptors to a specific compound possessing physiological activity. In addition, the invention provides proteins isolated and purified using the microspheres of the invention.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a microsphere which is preparedby coupling a substance possessing physiological activities to astyrene-glycidyl methacrylate polymer through a spacer as well as aprocess of isolating an objective or targeted substance by using themicrosphere of the invention.

[0003] 2. Background

[0004] Cells constituting a living body are exposed to various kinds ofstimulation from the external environment all the time. To respond tosuch stimulation the cells lead some gene groups to expression. As aresult, various living phenomena can occur, such as induction of cellgrowth and/or cell differentiation and maintenance of physiologicalhomeostasis. Extracellular stimulation is transformed into anintracellular signal, which activates a specific proteinoustranscription factor. The functionally activated transcription factorbinds to a specific base sequence on a chromosome to induce a gene groupunder its regulation to expression. The product of the induced geneexpression primarily functions to respond to the stimulation in somecases. In the other cases, the product of the induced gene expressionfurther activates another transcription factor that induces another genegroup under its regulation to expression to secondarily respond to thestimulation. In either case, cellular response to the stimulation fromthe external environment is concluded to be functional transformation oftranscription factors.

[0005] In recent years, an extremely interesting fact was revealed. Thatis, mechanisms of action of cyclosporin A (CysA) and FK506,immunosuppressive drugs, have been revealed. See J. Lin et al., Cell,66:807-815 (1991); S. J. O'Keefe et al., Nature, 357:692 (1992); and N.A. Clipstone et al., Nature, 357:695 (1992). The first opportunity forrevealing the mechanisms is the identification of intracellularreceptors to these drugs. See R. E. Handschumacher et al., Science, 226,554; and J. J. Sekierka et al., J. Immunol., 143:1580-1583 (1989). Onthe basis of these findings, a series of signaling pathway followingstimulation by antigen was revealed in T-cell that is immunocompetentcell.

[0006] Accordingly, investigation and identification of intracellularreceptors to drugs, as well as elucidation of signaling pathway, areexpected to be further developed into developmental research of newdrugs targeting the signaling pathway and research for novel drugdesigns.

[0007] Conventional methods of isolation and purification ofintracellular receptors to drugs are fractionation of crude cellextracts by using various columns, followed by detection of factorsbinding to labeled drugs in each fraction. Therefore, two steps ofprocedure, the first one was isolation and purification using columnsand the second one was assay for binding activity against drugs, havebeen necessarily performed until now.

[0008] Accordingly, for the purpose of purification, identification andfunctional analysis of receptors to a specific drug, located withincells or on cellular membrane, certain drug-immobilized microsphereshave been designed and constituted.

SUMMARY OF THE INVENTION

[0009] According to the conventional methods of purification, it cantake an exceedingly long time to purify drug-binding factors from crudecell extracts and moreover, a yield of factors is quite low due torepeated fractionation using various columns. Therefore, a huge amountof starting material is necessary for the determination of an amino acidsequence of drug-binding factors. It also can be most difficult toestablish an assay method for binding activity of receptors againstdrugs because obtained drug receptors are usually not identified.Conventional methods of determination of binding of receptors to drugsare filter binding method and gel filtration method which utilize thefact that drug receptors (proteins) bind to filters and that sizes ofdrugs binding to receptors become larger than free drugs and receptors.However, some receptors that do not bind to filters or other receptorschange their conformation after binding to filters and discharge drugs.Therefore, properties of drug receptors should be preliminarilyinvestigated in the conventional methods. The present invention aims atsolving the above mentioned problems to provide drug-immobilizedparticles and a process of purifying proteins.

[0010] According to the present invention, a microsphere comprisingstyrene-glycidyl methacrylate polymer is provided, and isolation,purification and identification of receptors to a specific compoundpossessing physiological activities are easily performed.

[0011] In addition, the present invention is concerned with microspheresprepared by coupling substances and proteins purified using themicrospheres of the invention.

[0012] The present invention is further concerned with microspherescomprising a substance possessing physiological activity, a polymer anda spacer, wherein at least one functional group of any of the substancepossessing physiological activity, the polymer or spacer is converted toanother type of functional group. The present invention also relates toa process of preparing such microspheres.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a schematic for the preparation method of astyrene-glycidyl methacrylate polymer and a styrene-glycidylmethacrylate polymer connected with a spacer.

[0014]FIG. 2 is a schematic for the isolation method of a protein usingmicrospheres of the present invention.

[0015]FIG. 3(A) shows the effects of E3330 on AP endonuclease activityof Ref-1. FIG. 3(B) shows the effects of NH₂-E3330 on AP endonucleaseactivity of Ref- 1.

[0016]FIG. 4 shows the enhancement of the DNA-binding activity ofNF-_(κ)B by recombinant rRef-1 and the repression of the recombinantrRef-1 activity by E3330.

[0017]FIG. 5 shows the effects of recombinant rRef-1 on the DNA-bindingactivities of r-p65 and/or r-p50.

[0018]FIG. 6 shows the procedure and the results of the GST pull-downassay.

[0019]FIG. 7 is a schematic for the structure of each recombinantprotein of Ref-1 expressed by various deletion mutants.

[0020]FIG. 8 shows the results of binding assays against E3330 using therecombinant proteins of Ref-1 expressed in E. coli by various deletionmutants.

[0021]FIG. 9 shows the results of binding assays against E3330 using therecombinant proteins of Ref-1 expressed in E. coli by various deletionmutants.

[0022]FIG. 10 shows the results of identification of E3330-bindingdomain using the recombinant proteins of Ref-1 expressed by variousdeletion mutants.

[0023]FIG. 11 shows the results of identification of E3330-bindingdomain using the recombinant proteins of Ref-1 expressed by variousdeletion mutants.

[0024]FIG. 12 shows the summary of E3330-binding domain in Ref-1identified using the recombinant proteins of Ref-1 expressed by variousdeletion mutants.

[0025]FIG. 13 shows reaction schemes for the conversion of epoxy groupsto other chemical groups.

[0026]FIG. 14 shows the isolation and purification of E3330-bindingproteins using E3330-immobilized particles.

[0027]FIG. 15 shows the isolation and purification of E3330-bindingproteins using E3330-immobilized particles.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides a microsphere prepared by couplinga compound possessing physiological activity to a styrene-glycidylmethacrylate polymer through a spacer. In addition, the presentinvention provides a process of isolating a substance by using amicrosphere prepared by coupling a compound possessing physiologicalactivities to a styrene-glycidyl methacrylate polymer through a spacer.

[0029] More particularly, the present invention is concerned with aprocess of isolating an objective substance that can adhere to asubstance possessing physiological activities from a mixture containingthe objective substance by using a microsphere prepared by coupling thesubstance possessing physiological activities to a styrene-glycidylmethacrylate polymer through a spacer. In more detail, the process ofisolating a substance according to the present invention includes mixingcrude cell extracts and microspheres prepared by coupling compoundspossessing physiological activity to styrene-glycidyl methacrylatepolymers through spacers, followed by isolation of microspheres, andthen, eluting the objective substance adhering to the substancespossessing physiological activities on the microspheres, to provide auseful process of isolating an objective or targeted substance.

[0030] The present invention also provides with proteins isolatedaccording to the above procedure. In addition, the present inventionprovides peptides or proteins comprising active moieties of isolatedproteins as receptors.

[0031] As the substance forming particles of microspheres in the presentinvention, styrene-glycidyl methacrylate polymers are employed. There isno special restriction on their states of polymerization ormorphological properties in the preparation method in which formedparticles should be isolated from a liquid phase. However, it ispreferable that particles are prepared by soap-free emulsionpolymerization according to the method developed by Inomata et al. suchas described in Y. Inomata et al., Anal. Biochem., 206:109 (1992).

[0032] The spacer in the present invention is a chemical compoundinterposed between the above particle and a compound possessingphysiological activity as discussed in more detail herein. A preferablespacer is a compound possessing one or more functional groups, such asan amino group, carboxyl group and epoxy group, on both its ends beforebinding to a particle and a substance possessing physiological activity.As for the selection of a spacer in the present invention, there is nospecial restriction but the spacer should be a substance connecting theabove styrene-glycidyl methacrylate polymer with a substance possessingphysiological activities at an appropriate distance. A particularlypreferred spacer is an ethylene glycol diglycidyl ether derivative.

[0033] As for the selection of a substance possessing physiologicalactivity (which will be occasionally abbreviated as physiologicallyactive substance, hereinafter) in the present invention, there is nospecial restriction but the substance should possess activity in aliving body and interaction and/or affinity to another substance ofintra- or extra-living body. It is further preferable to employ acompound that specifically binds to a receptor located within cells oron cellular membrane. In a case where the substance possessingphysiological activity has functional groups connectable with the abovespacer in its molecular structure, such as amino group and hydroxylgroup, the functional group is utilized as a group connected with aspacer. In the other case where the substance possessing physiologicalactivity has no functional group connectable with the above spacer inits molecular structure, a functional group connectable with the abovespacer is additionally induced into the concerned physiologically activesubstance and then, it is connected with a spacer. In either case it isnecessary to pay attention not to inactivate physiological activity ofthe concerned physiologically active substance by connecting thephysiologically active substance with a spacer. Namely, it is importantto confirm that an objective or desired physiological activity is notinactivated by connecting the physiological active substance with aspacer.

[0034] As the substance possessing physiological activities in thepresent invention, for example, a3-[(5-(2,3-dimethoxy-6-methyl-1,4-benzoquinonyl)]-2-nonyl-2-propionicacid (which will be occasionally abbreviated as E3330, hereinafter orderivative thereof) may be suitably employed. Other examples ofsubstances possessing physiological activity useful in the microspheresof the present invention include DM852, H-9, DQ2511 and KF49389.

[0035] A preparation method of a microsphere consisting of a compoundpossessing physiological activities coupled to a styrene-glycidylmethacrylate polymer through a spacer according to the present inventionis as follows. A particle composed of a styrene-glycidyl methacrylatepolymer (which will be occasionally abbreviated as a SG-particle,hereinafter) is prepared according to an ordinary method. However, forthe purpose of the SG-particle easily binding to a spacer, it isespecially preferable to prepare a particle with a functional glycidylgroup projecting on its surface and then, the glycidyl group on theSG-particle is ring-opened with appropriate reagents, such as ammoniumhydroxide, followed by induction of a preferable functional group forbinding to a spacer, depending on necessity. Subsequently, a spacer isbound to the SG-particle, followed by reaction with a substancepossessing physiological activities or its derivatives. Thus, amicrosphere of the present invention is prepared.

[0036] In these reactions, various solvents, such as dioxane, DMSO andwater, are suitably employed, depending on necessity.

[0037] In certain embodiments of the present invention, the functionalgroups on the components of the microsphere of the present invention,i.e., particle, spacer or physiologically active substances, areconverted to other types of groups having reactive carbons. For example,FIG. 13 shows the conversion of epoxide groups to other chemical groupsuseful for in the present invention. For the purposes of illustration,conversion of epoxide groups will be discussed. Epoxide groups on any ofthe spacer, particle or physiologically active substances can beconverted to another type of functional group. For example, an epoxidegroup can be covalently bound to a nucleophilic group such as hydroxy,amino and thiol group (reaction 1 in FIG. 13). In another embodiment,the epoxide group is treated with hydrochloric acid to introduce alkylchloride to the surface of the particle or the end of the spacer. Thechloride group can be substituted for a relatively strong nucleophile,such as thiol group (FIG. 13, reaction 2). This reaction is useful forselective incorporation of substances containing free thiol group. Thechloride group can be replaced by a good leaving group, such as ap-toluenesulfonyl group, which enables the reaction with weaknucleophilic groups.

[0038] In another example, the epoxide group is treated with ammonium,which enables the introduction of an amino group on the particle or onthe spacer. This amino group covalently binds to aldehyde groups viaSchiff base formation, and following reduction of the reaction siteresults in formation of a stable bond. (Reaction 3, FIG. 13). In yetanother example, the amino group covalently binds to a carboxyl group byusing a general coupling reagent for amide bond formation (reaction 4,FIG. 13). In yet another example, a carboxyl group is introduced byreacting succinic anhydride with the amino group. The generated carboxylgroup reacts with a hydroxy group or amino group by using a couplingreagent, such as N-hydroxysuccinimide, to form a stable ester bond oramide bond respectively (reaction 5).

[0039] These methods are useful in converting at least one functionalgroup on the particle, spacer, or physiologically active substances intoother types of activated groups. These resulting groups can then reactwith other components to form the microspheres of the present invention.For example, the epoxide group on glycidylmethacrylate, one ofcomponents of the preferred particles, is converted into other types ofactivated groups. Similarly, ethylenediglycidylether, one preferredspacer has an epoxide group at both ends. Either one, or both of theseepoxides can be converted into other activated groups. These convertedgroups are used to covalently bind to chemical groups, e.g., hydroxy,amino, thiol, aldehyde and/or carboxyl group contained inphysiologically activated substances.

[0040]FIG. 1 shows an example for the preparation of a microsphere ofthe present invention. Appropriate compounds, such as styrene andglycidyl methacrylate, are polymerized according to an ordinary method,such as emulsion polymerization, to prepare a SG-particle with afunctional glycidyl group projecting on its surface. Sizes of particlesare selectively varied according to circumstances. However, the sizes(lengths) are ordinarily about 0.05 to 0.5 μm and preferably they areabout 0.1 to 0.3 μm. The prepared SG-particle is reacted with a compoundemployed as a spacer, such as ethylene glycol diglycidyl ether (EDGE),to bind a spacer to the SG-particle. Thus, a spacer-binding SG-particle(SG-EGDE particle) is obtained. Then, the obtained particle is reactedwith a physiological active substance possessing a reactive functionalgroup, such as amino group, preferably in an organic solvent, such asdioxane. Thereby, a microsphere of the present invention, that is aphysiologically active substance-immobilized latex particle, isprepared.

[0041] As for the selection of a mixture containing an objectivesubstance for isolation according to the present invention, there is nospecial restriction but the mixture should contain a substancepossessing an affinity and selective binding ability to aphysiologically active substance employed in the preparation of amicrosphere. It is ordinarily preferable that cell extracts, especiallythe cell extracts from the concerned physiological activesubstance-acting sites are employed as a mixture.

[0042] The isolation procedure in the present invention is conducted asfollows. Physiologically active substance-binding microspheres and amixture containing proteins, such as cell extracts, are mixed andstirred, if necessary, for several minutes to several hours. Themicrospheres to which proteins are adhering are separated and rinsedwith a buffer solution, if necessary. Then, adhering proteins are elutedfrom the microspheres by using an appropriate solution, such as apotassium chloride solution, to be dissociated. Thus, the isolationprocedure is conducted. There is no special restriction on the states ofadhering conditions of an objective substance for isolation to aphysiological active substance employed in the preparation of amicrosphere. Any kinds of adhering, such as chemical bond (hydrogenbond, etc.) and chemical or physical adsorption, may be suitablyemployed.

[0043]FIG. 2 shows an example for the isolation of a protein in thepresent invention. In this example, E3330 is employed as aphysiologically active substance.

[0044] An obtained protein is detected by using SDS-PAGE or othersuitable method. In addition, an obtained protein is purified accordingto an ordinary method, such as chromatography, if necessary.

[0045] Proteins isolated according to a process of the present inventionare considered to be receptors to the physiologically active substanceof microspheres. However, proteins isolated according to a process inthe present invention are not limited to the receptors to thephysiological active substance employed in the preparation ofmicrospheres. Any kinds of substances possessing chemical, physical orbiological affinities to the physiological active substance employed inthe preparation of microspheres are able to be isolated according to aprocess in the present invention.

[0046] An obtained protein is purified, if necessary, and can besubjected to determination of its amino acid sequence. In addition,genes coding the concerned protein can be cloned according to anordinary biotechnological procedure, and their base sequences can bedetermined. Furthermore, according to an ordinary genetechnologyprocedure, proteins or peptides composed of partial amino acid sequencesof the concerned protein are expressed and an active moiety of thereceptor protein can be determined. These procedures are also describedin the working examples which follow, giving examples in which E3330 isemployed as a physiologically active substance.

[0047] Structures of obtained receptors or domains composed of activemoieties of the receptors can be elucidated through NMR analysis, x-raycrystal analysis or simulation analysis using computers on the basis oftheir amino acid sequences. For example, the protein, Ref-1, which isisolated according to a process of the present invention in which E3330is employed as a physiologically active substance in the preparation ofmicrospheres, is suggested to possess such structures asβ-sheet/α-helix/β-sheet in the domain of 82 a.a. to 106 a.a. through theabove structural analyses. The amino sequence of Ref-1 (SEQ ID NO:4) isset forth in Example 9 which follows. Regions of 72 to 88 amino acidresidues (SEQ ID NO:13) of Ref-1 are of particular interest and setforth in Example 14 which follows.

[0048] Further, peptides of the invention which have a sequence that ispartially deleted, added or substituted with respect to SEQ ID NO:4(which sequence is set forth in Example 9 which follows), and suchpeptides preferably comprise at least about 10 amino acids, morepreferably at least about 15 to 50 amino acids, still more preferably atleast about 40 to about 100 amino acids. Such peptides preferably haveat least about 70 percent homology (sequence identity) to SEQ ID NO:4,more preferably at least about 80 or 90 percent sequence homology to SEQID NO:4. Also, such preferred peptides preferably will contain a regionthat has substantial sequence identity (e.g. about 80, 90 or 95 percentor more sequence identity) to SEQ ID NO:13 (which sequence is set forthin Example 14 which follows).

[0049] On the basis of the results of such stereochemical structuralanalyses and genetic analyses, one can readily determine which aminoacid residue binds to a physiologically active substance employed in thepreparation of microspheres among amino acids of the proteins or activemoieties isolated according to a process of the present invention. Inaddition, one can investigate the interaction between the concernedphysiologically active substance employed in the preparation ofmicrospheres and amino acids of the isolated protein at molecular and/oratomic levels. Furthermore, it will be practicable to analyze chemicalkinetics of the binding reactions. Many findings obtained from the abovestudies will not only identify the protein that is the receptor to thephysiologically active substance employed in the preparation ofmicrospheres but also reveal the mechanism of action of the concernedphysiologically active substance in a living body. Moreover, it will bepracticable to conduct a novel drug design by accurately controlling anew drug at an atomic level, of which binding mechanism is differentfrom that of the physiological active substance employed in thepreparation of microspheres concerning the interaction with the proteinof the receptor. Various drugs designed according to the above methodreasonably possess different functions from those of the physiologicalactive substance employed in the preparation of microspheres. Therefore,the drugs will be utilized more properly for various purposes. Thus, aprocess in the present invention is extremely important for a novelprocedure of the drug designs.

[0050] In addition, the present invention provides a process forisolation and detection of substances possessing affinities to areceptor by employing a protein with an ability of the concernedreceptor as a substance possessing physiological activity coupled withmicrospheres. As a substance with an ability of a receptor, a wholeprotein of the receptor may be employed, but it is preferable to employa domain that is obtained by trimming the receptor protein to an activedomain of several tens (e.g. about 20 to 60) of amino acid residues asan active moiety of the concerned receptor.

[0051] Therefore, screening examinations on drugs specifically bindingto the concerned receptor or its active domain will be practicablyperformed, according to the above methods. Thus, chemical syntheticsubstances expected to be useful drugs are easily isolated and detectedamong various drug libraries by conducting the screening examinations.The substance isolated and detected through the above screeningexaminations should possess affinities to the protein with an ability ofa receptor coupled with microspheres. Therefore, the substance will bedeveloped to be an effective ingredient of a medicine for promotion orinhibition of the activity of the concerned receptor.

[0052] According to the present invention, a microsphere composed ofstyrene-glycidyl methacrylate polymer is provided, and purification andidentification of receptors to a specific compound possessingphysiological activities, located within cells or on cellular membrane,are easily performed. Particles coupled with a substance possessingphysiological activities specified in the present invention provideepoch-making and significant effects, that is, isolation andpurification of a receptor to a drug are able to be conductedsimultaneously with the evaluation on its binding activity to the drug,time required for isolation and purification of drugs is remarkablyshortened and a recovery ratio is extremely improved, and investigationon the assay method for binding activity against a drug is notnecessarily performed anymore. In addition, the present inventionprovides that an intracellular receptor to a drug or a compound can beisolated and purified by using the drug- or the compound-immobilizedparticles and then a structure and functions of the receptor can bedetermined. Furthermore, the present invention is indicated to beextremely useful for the development of novel drugs with superiorfunctions on the basis of many findings obtained from the structural andfunctional analyses on receptors to drugs according to a process in thepresent invention.

[0053] All documents mentioned herein are incorporated herein byreference. The present invention is further illustrated by the followingExamples. These Examples are provided to aid in the understanding of theinvention and are not to be construed as limitations thereof.

EXAMPLE 1 Preparation of Styrene-glycidyl Methacrylate Polymers

[0054] Styrene (St; Wako Pure Chemicals. It was used after distillationunder reduced pressure of 21.5 mmHg at 46° C.), glycidyl methacrylate(GMA; Wako 15 Pure Chemicals. It was used after distillation underreduced pressure of 2 mmHg at 33° C.), divinyl-benzene (DVB; TokyoKasei), 2,2′-azobis (2-amidinopropane dihydrochloride) (V-50; Wako PureChemicals) and water were used in the following compositional formula:St/GMA/DVB/V-50/H₂O=1.2/1.8+0.3/0.04/0.06/110 (g)

[0055] After substituting a nitrogen gas in the mixture, reaction ofpolymerization was conducted at 70° C. for 24 hours. To polymerize themixture soap-free emulsion polymerization was conducted according to themethod developed by Inomata et al. (Y. Inomata et al., Anal. Biochem.,206:109 (1992)).

[0056] Two hours after the initiation of polymerization, 0.3 g of GMAwas added to the mixture to thoroughly cover the whole surface of theobtained polymers with GMA. The obtained polymers (SG-particles) weresettled by centrifugation (15,000 rpm for 15 minutes at 4° C.), followedby decantation, and then re-suspended in 200 ml of water. The aboveprocedure was repeated three times to wash the SG particles and finallysuspended in water.

[0057] In order to induce amino groups into the washed SG-particles(0.25 g), NH₄OH (55.3 mmol; corresponding to fifty times larger amountof GMA unit) was added to the particles, followed by adjustment of thepH value to 11 with 1N HCl. The mixture was stirred using a stirrer at70° C. for 24 hours so that the epoxy group of GMA was ring-opened.

EXAMPLE 2 Immobilization of a Spacer

[0058] Next, an example for immobilization of ethylene glycol diglycidylether (EGDE; Wako Pure Chemicals), which was employed as a spacer, ontoSG-particles obtained in the working example 1 is shown in thefollowing.

[0059] An excess amount of EGDE, that was hundred times larger amount(mol) of amino groups on the surface of about 62.5 mg of SG-particlesprepared in Example 1 above, was added to the SG-particles and then, themixture was stirred at 30° C. for 24 hours at pH 11 (adjusted with 1NNaOH) so that an epoxy group of EGDE was covalently bound to an aminogroup on the surface of SG-particle. In order to avoid simultaneousimmobilization of two epoxy groups at the both ends of an EGDE moleculeonto SG-particles, such an excess amount of EGDE was added. Under thesereaction conditions about 1 mmol of EGDE was immobilized onto 1 g of theparticles. After the reaction was finished, the SG-particles were washedthree times with water through a centrifugation procedure. Thus theobtained spacer-immobilized particles, that are SG-EGDE particles, wereused for the particles to which a physiologically active compound isimmobilized.

EXAMPLE 3 Immobilization of an Amino Derivative of E3330 With a Spacer

[0060] (a) (Induction of an amino group into E3330)

[0061] As E3330 does not possess an appropriate functional group, it isdifficult to immobilize E3330 onto SG-EGDE particles. Therefore,NH₂-E3330 was synthesized through the induction of an amino group intoE3330.

[0062] (b) (Confirmation of the functions of NH₂-E3330)

[0063] The functions of NH₂-E3330 were compared with those of E3330 withrespect to transcriptional activation abilities of NF-_(κ)B. In order toexamine their functions transfection experiments were conducted byinducing the recombinant plasmid DNA possessing luciferase genesregulated by NF-_(κ)B into Jurkat cells. As a result, it was ascertainedthat NH₂-E3330 reduced not so strongly as E3330 but certainly reducedthe transcriptional activation abilities of NF-_(κ)B. Thus, it wasconfirmed that the amino group induced into E3330 did not inhibit thebinding of E3330 to intracellular receptors.

[0064] (c) (Immobilization of NH₂-E3330 to SG-EGDE particles)

[0065] Ten mg of SG-EGDE particles obtained in the working Example 2above was washed three times with 1 ml of 1,4-dioxane through acentrifugation procedure. After washing, 500 μl of 1,4-dioxane solutioncontaining 10 μmol of NH₂-E3330 was added to the packed SG-EGDEparticles to disperse the SG-EGDE particles in the above solution,followed by reaction at 37° C. for 24 hours, in order to immobilizeNH₂-E3330 to epoxy groups of EGDE on the surfaces of SG-EGDE particles.After the reaction was finished, the particles were washed three timeswith 20 μl of 1,4-dioxane through a centrifugation procedure. Then, theparticles were dispersed in 1 ml of 1M Tris-HCl buffer solution (pH7.4), allowed to be standing still at 4° C. for at least 24 hours andused, in order to thoroughly mask the intact epoxy groups on thesurfaces of SG-EGDE particles. The drug-immobilized particles werestored at 4° C. in a dark place. The centrifugation procedure forwashing was conducted at 15,000×g for 5 minutes at room temperature.Under these reaction conditions about 0.15 mmol of NH₂-E3330 wasimmobilized onto 1 g of the SG-EGDE particles. The above immobilizedamount of NH₂-E3330 was obtained by subtracting the amount of NH₂-E3330not bound to the SG-EGDE particles from the starting amount ofNH₂-E3330. NH₂-E3330 shows the maximum absorption at the wavelength of254.5 nm, so that each amount of NH₂-E3330 can be determined bymeasuring an absorbance at the wavelength of 254.5 nm on each sample,such as the starting solution, not-binding fraction and washingfractions. The measurement on the absorbance was conducted with DU-64Spectrophotometer (BECKMAN).

EXAMPLE 4 Preparation of a Crude Nuclear Extract and a CytoplasmicFraction

[0066] The culture medium suspension of Jurkat cells (2×10¹⁰ cells),which were cultured in a suspension scale of 8 liters, was centrifugedusing 500-ml-centrifugation tubes (NARGEN) at 500×g for 10 minutes at 4°C. for the purpose of collecting the cells. The collected cells werewashed two times with PBS(−). The washing procedure was conducted using50-ml-centrifugation tubes and the centrifugation conditions were at700×g for 5 minutes at 4° C. Then, the final packed cell volume (PCV)was measured. Buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5mM DTT), four times larger volume of the PCV, was added to the packedcells to suspend the cells. The cell suspension was allowed to standstill on ice for 20 minutes so that the cells were swollen. The cellmembranes of the swollen cells were broken by 20 strokes using a40-ml-B-type Dounce homogenizer (WHEATON), transferred to a50-ml-centrifugation tube (NARGEN) and centrifuged at 4,200×g for 6minutes at 4° C. for the purpose of separating a nuclear fraction(pellet) from a cytoplasmic fraction (supernatant).

[0067] Buffer A, five times larger volume of the PCV, was added to theisolated nuclear fraction to re-suspend the nuclei. The nuclearsuspension was centrifuged at 4,200×g for 6 minutes at 4° C. for thepurpose of removing the contaminated cytoplasmic fraction. The obtainednuclear pellet was dispersed in Buffer C (20 mM Hepes pH 7.9, 25% (v/v)glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM PMSF, 1 mMDTT), the same volume as the PCV, and thoroughly suspended by 10 strokesusing a B-type Dounce homogenizer. The suspension was slowly stirred for30 minutes at 4° C. for the purpose of extracting nuclear components.The extract was transferred to a 50-ml-centrifugation tube andcentrifuged at 16,000×g for 30 minutes at 4° C. The obtained supernatantwas dialyzed two times against one liter of Buffer D (20 M Hepes pH 7.9,20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 1 mM DTT) for2.5 hours at 4° C.

[0068] On the other hand, the cytoplasmic fraction was transferred toanother 50-ml-centrifugation tube and centrifuged again at 4,200×g for 6minutes at 4° C. The obtained supernatant was transferred to anultra-centrifugation tube (BECKMAN: No. 355620) and ultracentrifuged at35 Krpm for one hour at 4° C. (BECKMAN: Rotor Type 35). The obtainedsupernatant was dialyzed in the same manner as the above procedure forthe nuclear extract.

[0069] After the completion of the dialyses, the nuclear extract and thecytoplasmic fraction were centrifuged at 14,000×g for 30 minutes at 4°C. The obtained supernatants were used as the samples of a nuclearextract and a cytoplasmic fraction, respectively. These samples weresubdivided into appropriate aliquots and stored at −80° C. until the useof them.

[0070] In usual preparation, about 20 ml each of the nuclear extract andthe cytoplasmic fraction at the protein concentration of 5 mg/ml and 10mg/ml, respectively, were obtained in the scale of this working example.

EXAMPLE 5 Fractionation of a Nuclear Fraction and Cytoplasmic FractionUsing Phosphocellulose

[0071] Cationic-ion exchange phosphocellulose (P11: Whatman) wasemployed for the fractionation of a cell extract. Procedures for thefractionation were always conducted at 4° C. Distilled water was addedto 10 g of dried phosphocellulose to be 500 ml of a phosphocellulosesuspension. The suspension was well stirred and allowed to stand stillfor 30 minutes. To remove phosphocellulose with smaller particle sizesthe supernatant of the suspension was removed and distilled water of thesame volume as the removed supernatant was added to the remainingphosphocellulose.

[0072] These procedures were repeated several times in order to obtainthe Phosphocellulose with a uniform particle size. Next, in order toactivate the Phosphocellulose, each one gram of the phosphocellulosewith a uniform particle size on the basis of the dried weight wassuspended in 50 ml of 0.5 N HCl solution and the suspension was allowedto stand for 5 minutes. The phosphocellulose was collected on two sheetsof filter paper (Whatman: 3MM Chr) placed in Buchner funnel and washedwith an excess amount of distilled water until the pH value recovered toa neutral range. The pH value of the filtrate was measured using BTB pHtest paper to confirm the pH value. The phosphocellulose on the filterpaper was transferred into a beaker and resuspended in 0.5 N NaOHsolution. The suspension was allowed to stand for 5 minutes. Thephosphocellulose was washed in the same manner as described above.Finally, the phosphocellulose was suspended in 0.5 N HCl solution oncemore and washed again. As a result, activated phosphocellulose wasobtained.

[0073] The activated phosphocellulose was filled into a column (BIORAD:731-1550), so that the column volume became 10 ml. The filled column waswashed with 100 ml of Buffer D (20 mM HEPES (pH 7.9), 20% (v/v)glycerol, 1 M KCl, 1.2 mM EDTA, 0.5 mM PMSF, 1 mM DTT) containing 1 NKCl and then equilibrated with 100 ml of Buffer D containing 0.1 M KCl.Each 5 ml of a nuclear extract and a cytoplasmic fraction from Jarkatcells was applied on the column. The applied column was eluted stepwisewith Buffer D at various salt concentrations of KCl, that is, theelution was conducted stepwise with Buffer D containing 0.1 M KCl, 0.32M KCl, 0.35 M KCl or 1 M KCl. Each eluted fraction was dialyzed againstBuffer D, subdivided into appropriate aliquots and stored at −80° C.until the use of them.

[0074] The flow rate was adjusted to be 8 ml/minute. To confirm thecompletion of each step of protein-elution with each salt concentrationbuffer, absorbance at the wave-length of 280 nm was measured with UVdetector (GILSON: MODEL 111B) during the fractionation.

[0075] First, 5 ml of a cytoplasmic fraction (protein concentration: 12mg/ml) as applied on the above column. As a result, 30 ml of 0.1 Mfraction (P.1; protein concentration: 1.12 mg/ml), 39 ml of 0.3 Mfraction (P.3; protein concentration: 0.23 mg/ml), 37 ml of 0.5 Mfraction (P.5; protein concentration: 0.07 mg/ml), and 53 ml of 1.0 Mfraction (P1.0; protein concentration: 0.02 mg/ml) were obtained.

[0076] Next, a nuclear extract fraction was also fractionated using thephosphocellulose column in the same manner.

EXAMPLE 6 Isolation of Proteins Using Microspheres

[0077] A process of isolation and purification of E3330-binding proteinsusing E3330-immobilized particles is illustrated in the FIG. 2 anddescribed as follows.

[0078] (a) Microspheres and a fraction obtained through thefractionation using a phosphocellulose column in working Example 5 abovewere mixed and centrifuged to separate substances binding to E3330 whichwas immobilized on particles from the mixture. The centrifugationprocedure for separation was conducted at 15,000×g for 5 minutes at 4°C. All procedures in the above were conducted at 4° C.

[0079] (b) First, 1 mg each of E3330-not-immobilized SG-EGDE particlesand E3330-immobilized SG-EGDE particles were washed three times with 400μl of Buffer D′ (20 mM HEPES (pH 7.9), 10% (v/v) glycerol, 0.1 M KCl,0.2 mM EDTA, 1 mM DTT) in which glycerol concentration was 10% insteadof 20% in Buffer D. The E3330-not-immobilized SG-EGDE particles weredispersed in 1 ml of 1 M Tris-HCl buffer solution (pH 7.4) and allowedto stand still at 4° C. for at least 24 hours in order to mask epoxygroups of EGDE. These particles were used as a reference control againstE3330-immobilized SG-EGDE particles. To these washedE3330-not-immobilized SG-EGDE particles and E3330-immobilized SG-EGDEparticles, 200 μl each of P.1, P.3, P.5 or P1.0, which were obtainedthrough the fractionation of a cytoplasmic fraction using aphosphocellulose column, was added and mixed. These mixtures wereallowed to stand still for 30 minutes with intermittently stirring atintervals of 10 minutes in order to bind proteins possessingE3330-binding abilities to E3330 which was immobilized on the particles.The mixture was centrifuged and the supernatant was discarded. Thepellet was washed three times with 500 μl of Buffer D′ to removenon-specific binding substances as much as possible.

[0080] Subsequently, the washed pellet was eluted three times withBuffer D′ containing 50 μl of 1 M KCl, so that the proteins possessingE3330-binding abilities were dissociated and eluted from E3330immobilized on the particles. The wash solution and eluate solution werestored at −80° C.

[0081] (c) The detection of the proteins possessing E3330-bindingabilities was conducted by electrophoresis on a 10% SDS-polyacrylamidegel (SDS-PAGE) using 25 μl each of the first, second or third eluatesolution obtained from the E3330-immobilized SG-EGDE particles andE3330-not-immobilized SG-EGDE particles used as a reference control. Theproteins possessing E3330-binding abilities were collected with morethan 70% yield in the first elution and further collected with more than90% yield in the first and second elutions. In this experiment, 4×SDSsample dye (200 mM Tris-HCl (pH 6.8), 500 mM β-mercaptoethanol (β-ME),8% SDS, 0.4% BPB) was used instead of 4×SDS sample dye in order toprevent the electrophoresis from being disordered due to such highconcentration of the salts. The electrophoresed gel was subjected tosilver staining and the proteins specifically binding to E3330 wereidentified in comparison with the results of the reference control. As aresult, in the P.5 fraction a protein band with a molecular weight ofabout 38 kDa which was not observed in the reference control was clearlyobserved, suggesting that the protein was specifically binding to E3330.Concerning the other fractions, there were no significant differences inthe protein bands of the eluate solution with 1 M KCl, compared with theresults of the reference control.

[0082] The above procedure was repeated and finally 5 μg ofE3330-binding protein was obtained.

EXAMPLE 7 Evaluation of Specific-binding Abilities Against E3330

[0083] Two kinds of experiments were conducted to confirm that theprotein with a molecular weight of about 38 kDa in the P.5 fractionsfrom cytoplasmic fractions and nuclear extracts of Jurkat cells wasspecifically bound to E3330.

[0084] The first one was a competitive binding-inhibitory experiment.When in the step of a procedure for the addition of P.5 fraction of thecytoplasmic fraction fractionated using the phosphocellulose (200 μl) toSG-EGDE particles free E3330 at the same moles of the NH₂-E3330immobilized on the particles or free NH₂-E3330 at ten times more molesthan the immobilized NH₂-E3330 were added simultaneously, the proteinpossessing specifically binding abilities to E3330 immobilized on theparticles would be bound to free E3330 or free NH₂-E3330, resulting in alower yield in the isolation using the particles. As E3330 was insolublein water, E3330 was dissolved in EtOH, diluted with Buffer D and thenadded to the particles (the final concentration of EtOH was 2%). As aresult, it was confirmed that the yield of the protein with a molecularweight of about 38 kDa was lowered, indicating that the protein wasspecifically bound to E3330.

EXAMPLE 8

[0085] Next, the other experiment was conducted by varying the amount ofNH₂-E3330 immobilized on the SG-EGDE particles. In the present study themaximum amount of the immobilized E3330 derivatives is 0.4 μmol per 1 mgof SG-EGDE particles. Under these conditions, about 5 to 6 molecules ofE3330 derivatives are immobilized on the 1 mm² of the surface of theparticles. This experimental study was conducted in case where theamount of the immobilized E3330 derivatives was 0.2 μmol or 0.4 μmol per1 mg of SG-EGDE particles. However, in the present invention, amounts ofcompounds immobilized on the particles are varied depending on theproperties of the immobilized compounds, conditions of immobilizationand so on. The amounts are not defined and are generally varied betweena few molecules and hundred molecules. As a result, it was confirmedthat the yield of the protein with a molecular weight of about 38 kDaincreased as the immobilized amount increased. The identification of thespecific protein was conducted by electrophoresis using SDS-PAGE.

EXAMPLE 9 Determination of Amino Acid Sequence of E3330-binding Protein

[0086] The obtained E3330-binding protein was dialyzed two times againstone liter of Buffer D for 2.5 hours at 4° C. in order to remove thecontaminated KCl that was used as an eluent solution of such as highconcentration of 1.0 M of KCl. In order to concentrate the sample, afterthe dialysis was completed, the dialyzed sample was transferred into anultra-centrifugation tube (BECKMAN: No. 331372) to which trichloroaceticacid (TCA: MERCK) and deoxycholic acid (DOC: Sigma) were added so thatthe final concentrations would be 10% and 0.8 mg/ml, respectively. Themixture was stirred well and allowed to stand still for 30 minutes onice. Then, the mixed sample was ultra-centrifuged at 28 krpm, for 15minutes at 4° C. (BECKMAN: Rotor SW 41 Ti). The obtained precipitationwas dissolved in 10 ml of acetone. After standing still for 10 minutesat room temperature, the solution was ultra-centrifuged again. Theobtained precipitation was allowed to stand still for 10 minutes on iceto be dried. Thus, the sample was concentrated.

[0087] Finally, the concentrated sample was dissolved in 50 μl of 1×SDSsample dye and transferred into a sample tube (Eppendorf: No. 0030102.002). In this procedure, the remaining sample in theultracentrifugation tube was rinsed with additional 10 μl of 1×SDSsample dye and pooled in order to collect the remaining sample.

[0088] As TCA and DOC were remaining in the sample, 3 μl of 1 M Tris-HCl(pH 7.9) was added for the purpose of neutralization. Then, the samplewas stored at −80° C.

[0089] To analyze the amino acid sequence of E3330-binding protein, theprotein was subjected to peptide-fragmentation. First, the sample waselectrophoresed with pre-stained SDS-PAGE standard (BIO-RAD) on a 10%SDS-polyacrylamide gel and the protein was transferred into PVDFmembrane (MILLIPORE: immobilon transfer membrane) from thepolyacrylamide gel using Mini trans blott module kit (BIO-RAD). Prior tothe blotting, the PVDF membrane was soaked in methanol for 15 secondsand subsequently it was soaked in a blotting buffer solution (10 mMCAPS-NaOH (pH 11), 10% methanol) for more than 5 minutes. The blottingapparatus was placed on the anode side down and two sheets of fiber pad,two sheets of 3 MM paper, PVDF membrane, the gel, two sheets of 3 MMpaper and two sheets of fiber pad were laid in the blotting apparatus inthat order, avoiding bubbles. The apparatus was placed in anelectrophoretic bath filled with a blotting buffer solution. The bathwas being chilled with ice and 0.3 A of electric current was turned onfor 30 minutes to blot the protein into PVDF membrane.

[0090] In case where the proteins blotted on the membrane wereenzymatically digested, Lysil Endopeptidase (Wako Pure Chemicals) wasemployed as the digestive enzyme. As a buffer solution for the digestivereaction 20 mM Tris-HCl (pH 8.8) with 8% acetonitrile was used. Theamount of the enzyme was one tenth of the protein for the digestion(g/g). First, a half amount of the enzyme was added, followed bystirring with shaking for several seconds. Then, the other half of theenzyme was added to the above, followed by stirring with shaking at 37°C. for about 24 hours under prevention of light-transmittance. After thecompletion of the reaction, the digested fluid was carefully collected,paying attention to the PVDF membrane not to be sucked up. The remainingPVDF membrane was washed with 100 μl of 8% acetonitrile. The washingfluid was collected in the same manner as the above and pooled. Thesefluids were centrifuged at 15,000×g for 2 minutes at room temperature inorder to completely separate the contaminated PVDF membrane pieces andremove them. In case where the supernatant is directly applied on highperformance liquid chromatography (HPLC), highly hydrophilic peptidesare eluted in a passing through fraction. Therefore, the supernatant wasconcentrated by decompression so that the concentration of acetonitrilewas reduced. To the concentrated supernatant 0.1% trifluoroacetic acid(TFA) was added so that the volume became 205 μl, which was applied onreversed phase HPLC (ABI: model 130A). C8 column (PERKIN ELMER:0711-0056) was employed. Flow rate was 50 μl/min and column temperaturewas 35° C. for development. Elution was conducted as follows; mobilephase was 0.1% TFA and concentration gradient of acetonitrile in themobile phase was 0% for the first 5 minutes, 0 to 35% for the next 30minutes and 35 to 70% for the last 20 minutes. Monitoring protein wasconducted using ultraviolet absorption at the wave-length of 215 nm.Each eluted peptide fraction was collected at each time and stored at−80° C.

[0091] Gas phase protein sequencer (ABI: model 477A protein sequencer)was used for amino acid sequence analysis on the peptides. Polybrene(ABI) was used as the carrier. As a result, three amino acid sequencesof peptides were determined. The obtained sequences were GLDWVK (SEQ IDNO:1)/AAGEGPALYEDPPD (SEQ ID NO:2)/GAVAEDGDEL (SEQ ID NO:3). These aminoacid sequences were analyzed using a computer and determined to becompletely identical with the amino acid sequences of N-terminalflanking region of redox protein, Ref-1 which participates inoxidation-reduction reaction. The Ref-1 has been reported to possess 318amino acid residues with a molecular weight of 38 kDa. The proteinobtained through the isolation and purification in this working examplepossesses the same molecular weight. Therefore, E3330-binding protein isprobably identical with Ref-1. Amino acid sequence of Ref-1 is asfollows (SEQ ID NO:4 length of sequence 318 amino acids):MPKRGKKGAVAEDGDELRTEPEAKKSKTAA 1        10        20       30KKNDKEAAGEGPALYEDPPDQKTSPSGKPA 31       40        50       60TLKICSWNVDGLRAWIKKKGLDWVKEEAPD 61       70        80       90ILCLQETKCSENKLPAELQELPGLSHQYWS 91      100       110      120APSDKEGYSGVGLLSRQCPLKVSYGIGDEE 121     130       140      150HDQEGRVIVAEFDSFVLVTAYVPNAGRGLV 151     160       170      180RLEYRQRWDEAFRKFLKGLASRKPLVLCGD 181     190       200      210LNVAHEEIDLRNPKGNKKNAGFTPQERQGF 211     220       230      240GELLQAVPLADSFRHLYPNTPYAYTFWTYM 241     250       280      270MNARSKNVGWRLDYFLLSHSLLPALCDSKI 271     280       290      300RSKALGSDHCPITLYLAL 301     310    318

EXAMPLE 10 Production of E3330-binding Protein by Gene Recombination

[0092] (a) The procedures for preparation of cDNA clones ofE3330-binding protein and analysis on the binding activity of theprotein produced through gene expression of recombinant cDNA clones inEscherichia coli (E. coli) against E3330 were conducted as follows.

[0093] On the basis of the determined amino acid sequences E3330-bindingfactor is considered most probably to be Ref-1. Therefore, cDNA clonesof Ref-1 were prepared and the recombinant cDNA clones were expressed inE. coli to obtain recombinant proteins. Then, the binding activity ofthe protein against E3330 was investigated.

[0094] (b) First, RNA was prepared. When RNA was prepared,ultra-centrifugation tubes (BECKMAN: 331372), bucket (BECKMAN: for SW41Ti) and bucket cap (BECKMAN: for SW 41Ti) were preliminarily soaked in2% absolve solution (DUPONT; 20 ml of absolve was diluted with distilledwater to be 1000 ml) on the day before the preparation to removecontaminated RNase activities as much as possible. They were thoroughlyrinsed with distilled water just before the use. Cultivation medium ofJurkat cells cultured up to 4.2 liter (7.6×10⁹ cells) was transferred to500-ml-centrifugation tubes and centrifuged at 500×g for 5 minutes at 4°C. to collect the cells. The collected cells were washed two times withPBS(−). The centrifugation conditions for washing were 700×g for 5minutes at 4° C. using 50-ml-centrifugation tubes. At that time packedcell volume (PCV) was simultaneously measured.

[0095] These cells were thoroughly suspended in 10-fold PCV of guanidiumsolution (4 M guanidium thiocyanate, 0.1 M Tris-HCl pH 7.5, 1% (v/v)β-ME). The suspension was passed through an injection needle of 18G (1.2mm) (TERUMO: NN-1838R) twenty times and further through an injectionneedle of 25G (0.5 mm) (TERUMO: SS-20ES) to cut DNA strands intofragments. To the resultant suspension 10% N-lauroylsarcosine was addedand thoroughly stirred, so that the final concentration oflauroylsarcosine became 0.5%. Then, 3 ml of each aliquot of the mixturewas carefully placed over the phase of 9 ml of CsCl/EDTA solution (5.7 MCsCl, 0.01 M EDTA pH 7.5) which was preliminarily poured into anultra-centrifugation tube. The ultracentrifugation tubes were placed inthe bucket and bucket cap was fastened. Then, the bucket was fixed in arotor (SW 41Ti) and ultracentrifuged at 32 kprm for 24 hours at 20° C.

[0096] The supernatant was carefully discarded thoroughly and the upperpart of the ultra-centrifugation tube was cut of f by a cutter. Theobtained precipitation was washed with 70% ethanol. Then, the pellet wasrinsed three times with 150 μl of TE/SDS solution (10 mM Tris-HCl pH7.6, 1 mM EDTA, 0.1% SDS) to dissolve the precipitation in TE/SDSsolution. The solution was extracted two times with phenol/chloroform,followed by addition of 900 μl of ethanol and 30 μl of 3 M sodiumacetate (pH 5.2). The mixture was stored at −80° C. until use. When theconcentration was measured, an aliquot of the above stock solution wastaken out and centrifuged (15,000×g for 5 minutes at 4° C.). Afterwashing with 70% ethanol (15,000×g for 5 minutes at 4° C.), theprecipitation was dissolved in water that was pretreated with diethylpyrocarbonate (DEPC) (DEPC was added to distilled water so that thefinal concentration of DEPC became 0.1%, followed by stirring, standingstill for about 24 hours and autoclaving. The pretreated water wasstored at room temperature.). Absorbance of the solution was determinedat the wave-length of 260 nm using DU-64 Spectrophotometer and theamount of RNA was estimated.

[0097] (c) Next, complementary single-stranded cDNA was prepared byreverse transcription. An aliquot of RNA (10 μg) obtained in the aboveprocedure was taken out and centrifuged (15,000×g for 5 minutes at 4°C.). The obtained precipitation was washed with 70% ethanol (15,000×gfor 5 minutes at 4° C.) and dissolved in 9.8 μl of DEPC-treated water.To the solution 160 ng of 0.5 μg/μl Oligo(dT)¹⁵ primer (Promega:5′-TTTTTTTTTTTTTTT-3′) was added and heated at 70° C. for 5 minutes.After the heating for 5 minutes, the sample was promptly placed on iceand Preliminarily ice-chilled 28 μl of reaction buffer (a mixture ofReverse Transcriptase attachments, that is, 8 μl of 5×RT Buffer, 4 μl of0.1 M DTT, 2 μl of 10 mM dNTPs, and 13 μl of distilled water), 1 μl ofRibonuclease inhibitor (TaKaRa: Ribonuclease inhibitor) and 2 μl ofReverse transcriptase (GIBCO BRL: Super ScriptTM RNase H-ReverseTranscriptase) were added to the sample in this order. Then, the mixedsample was promptly subjected to one-hour reaction at 37 C to elongatethe complementary single-stranded cDNA by reverse transcription. At theend of the reaction, the sample was heated at 95° C. for 5 minutes tostop the reaction by heat block. The obtained complementarysingle-stranded cDNA was stored at −30° C. until use.

[0098] (d) Subsequently, oligonucleotides were synthesized for thepurpose of amplification of Ref-1 translational region by Long-PCRmethod. Each base sequence of the synthesized oligonucleotides is shownin the following. Each oligonucleotide possesses individual digestiveregion by the restriction enzyme from which each oligonucleotides takesits name. 5′Ref-1XhoI primer: 5′-GTCTCTCGAGATGCCGAAGCGTGGGAAAAAG-3′ (SEQID NO: 5) 3′Ref-1 BamHI primer: 5′-ATGCGGATCCTTACAGTGCTAGGTATAGGGT-3′(SEQ ID NO: 6)

[0099] The synthesized oligonucleotides were heated at 55° C. for 8hours to be deprotected. The deprotected oligonucleotides weresubdivided into aliquots, dried under vacuum and dissolved in diluted (1in 10) buffer of TE (10 mM Tris-HCl pH 7.9, 1 mM EDTE). PCR wasconducted with the above two kinds of oligonucleotides, employing theprepared single-stranded cDNA as templates. PCR kit (XL PCR kit: PERKINELMER) was employed for the PCR procedures. There are two kinds ofreaction solutions (Lower Layer and Upper Layer). The Lower Layercontains 40 pmol each of S′-terminal primer and 3′-terminal primer,dNTPs of final concentration of 0.8 mM, Mg(OAc)2 of final concentrationof 1.4 mM and 12 μl of 3.3×XL Buffer II. The final volume was made to be40 μl. On the other hand, the Upper Layer contains 1 μl of thesingle-stranded CDNA templates, rTth DNA Polymerase, XL 4U and 18 μl of3.3×XL Buffer II. The final volume was made to be 60 μl.

[0100] First, GEM 100 WAX (PERKIN ELMER) was placed on the Lower Layerof a sample tube and heated at 80° C. for 5 minutes using a geneamplification apparatus, followed by cooling at 25° C. for one minute,so that the WAX was solidified on the Lower Layer. Thereon Upper Layerwas placed and subjected to the reaction using a gene amplificationapparatus according to the following scheme; at 94° C. for one minute,16 cycles of (at 94° C. for 15 seconds, at 60° C. for 10 minutes), 12cycles of (at 94° C. for 15 seconds, at 600° C. for 10 minutes(elongated by 15 seconds in every cycle)), and at 72° C. for 10 minutes.After the completion of the reaction, the WAX which was solidified onthe Upper Layer was holed through and the reaction solution wastransferred, followed by chloroform extraction and ethanolprecipitation. The amplified DNA fragments were digested by XhoI andBamHI (TOYOBO) and directly subjected to agarose gel electrophoresis. Apart of the agarose gel containing the DNA fragments was isolated,followed by phenol/chloroform extraction and ethanol precipitation topurify the DNA fragments.

[0101] (e) As the E. coli expression vector, pET14b (Novagen) wasemployed. The DNA fragments purified in the above procedure were ligatedwith the isolated and purified pET14b XhoI/BamHI-digested fragments inorder to construct the E. coli expression plasmids (pET/Ref) which wouldexpress the recombinant protein of Ref-1 wild type.

[0102] The E. coli possessing pET14b-derived E. coli expression plasmidsexpresses His-Tag fused recombinant protein of which N-terminal regionis a peptide consisting of six histidines.

EXAMPLE 11 Confirmation of the Binding Ability of Ref-1 Against E3330

[0103] (a) The binding ability of the recombinant protein obtained inthe above procedure against E3330 was investigated usingE3330-immobilized particles. As a result, it was certainly confirmedthat the recombinant protein of Ref-1 specifically bound to E3330.Furthermore, it was confirmed that E3330 bound to Ref-1 in Far Westernmethod using ¹⁴C-labeled E3330. Therefore, Ref-1 is regarded asintracellular receptor to E3330.

[0104] (b) Ref-1 consists of a domain possessing redox activity in itsN-terminal region and consists of another domain possessing AP nucleaseactivity, that severs apurinic/apyrimidinic single-stranded DNA andinserts nick, in its C-terminal region. Therefore, it was investigatedwhether E3330 would inhibit these activities or not in the nextexperiment.

[0105] First, concerning AP nuclease activity, the plasmid pBluescriptSK+DNA (50 μg) was treated with 50 mM sodium citrate (pH 3.5) at 60° C.for 15 minutes, followed by dialysis in 50 mM Tris-HCl (pH 7.4) at 4° C.for about 24 hours. This AP plasmid DNA possesses supercoiled circularDNA structure. This DNA was suspended in nuclease buffer solution (10 mMTris-HCl (pH 8.0), 5 mM MgCl₂, 1 mM EDTA, 0.01% NP-40), to which therecombinant Ref-1 was added, resulting in insertion of nick and opencircular DNA structure. However, as shown in FIG. 3, the AP nucleaseactivity of the Ref-1 was not inhibited by E3330.

[0106] (c) Next, effects of Ref-1 on a redox activity were investigated.

[0107] The obtained results are shown in FIG. 4. Prior to the conductionof the investigation, there are some considerations. Transcriptionfactor NF-_(κ)B possesses plural cystine residues in its amino acidsequence. There are two cases; one is an oxidized condition where thesecystine residues are bound to each other through an S—S bond; and theother one is a reduced condition where they are individually existingwith an SH-group. Therefore, NF-_(κ)B was treated with dithiothreitol(DTT) that is known as a reductant to make the state of NF-_(κ)B to bereduction. As a result, DNA-binding ability of the reduced NF-_(κ)B wasincreased, that was confirmed by a gel-shift assay. Ref-1 possessing aredox activity was added to NF-_(κ)B which was partially purified fromJurkat cells, resulting in increase in DNA-binding ability, that wasconfirmed by a gel-shift assay. Furthermore, the enhancement of theDNA-binding activity of NF-_(κ)B by Ref-1 was reduced by the addition ofE3330 to the reaction mixture. However, E3330 did not inhibit theincrease in the DNA-binding activity of NF-_(κ)B by DTT. Therefore, itwas revealed that E3330 specifically inhibits the DNA-binding activityof NF-_(κ)B.

[0108] (d) NF-_(κ)B is a hetero-dimer consisting of two subunits (p65and p50). The molecular weights of those two subunits p65 and p50 are 65kDa and 50 kDa, respectively. The His-tag recombinant proteins of thosep65 and p50 subunits were prepared using baculovirus expression system.Then, it was investigated by gel-shift assay whether E3330 had effectson p65 or on p50. The obtained results are shown in FIG. 5. DNA-bindingability of p65/p65 homo-dimer under reduced conditions by the additionof DTT was significantly enhanced, but the enhancement was not observedby the addition of Ref-1. On the other hand, DNA-binding ability ofp50/p50 homo-dimer and p65/p50 hetero-dimer was enhanced by the additionof either DTT or recombinant Ref-1. Therefore, it was revealed thatRef-1 had effects on p50, one of the subunits NF-_(κ)B. The enhancementof DNA-binding ability of p50/p50 homo-dimer or p65/p50 hetero-dimer bythe addition of Ref-1 was reduced by the addition of E3330. As NF-_(κ)Brecognizes a specific base sequence and binds to the specific DNAsequence, binding to DNA is the minimum requirement for NF-_(κ)B tofunction as a transcriptional factor. This binding step is regulated byRef-1, indicating that Ref-1 is at least an intracellular factoractivating transcription factor, NF-_(κ)B with respect to DNA-bindingstep. Moreover, it was revealed that E3330 inhibits the activationinduced by Ref-1.

[0109] (e) In addition, GST pull down assay was conducted to confirmthat Ref-1 specifically bound to p50 subunit of NF-_(κ)B. The obtainedresults show that Ref-1 really binds to p50 of GST-tag (GST-p50), asindicated in FIG. 6.

EXAMPLE 12 Preparation of Mutational Recombinant Proteins of Ref-1

[0110] (a) In order to confirm that Ref-1 bound to E3330, a series ofdifferent quantities of N-terminal and C-terminal flanking regions ofRef-1 deletion mutant strains were prepared using pET/Ref, as describedin the following. Then, these recombinant proteins were expressed in E.coli and purified using a nickel column and a glutathione column toinvestigate whether they bound to E3330-immobilized particles or not.These mutational recombinant proteins of Ref-1 including a wild type areschematically shown in FIG. 7.

[0111] (b) Preparation of pET/RefdC76, pET/RefdC91, pET/RefdC163 andpET/RefdC 182

[0112] The prepared pET/Ref, 10 μg, was digested with BamHI and AatII(TOYOBO), extracted with phenol/chloroform and precipitated withethanol. These pET/Ref BamHI/AatII digested fragments were dissolved in100 μl of the buffer solution which was prepared through dilution (1 in10) of 10×ExoIII Buffer (500 mM Tris-HCl (pH 8), 50 mM MgCl₂, 100 mMβ-ME) attached to Exonuclease III (ExoIII: TaKaRa). Subsequently, 180 Uof ExoIII was added to the above solution, followed by incubation at 25°C. for 5, 10, 15, 20, 25, 30, 40, 50, or 60 minutes, for the purpose ofdegradation of the pET/Ref BamHI/AatII digested fragments in thedirection of 3 to 5′. Since ExoI is inhibited by the addition of Zn+,100 μl of the buffer solution which was prepared through dilution (1 in10) of 10×Mung Been Buffer (300 mM CH₃COONa (pH 4.6), 1 M NaCl, 10 mM(CH₃COO)₂Zn, 50% Glycerol) attached to Mung Been Nuclease (TaKaRa) wasadded to each of the reaction mixtures to stop the degradation. Theresultant reaction mixtures were incubated at 65° C. for 5 minutes toinactivate the enzyme completely. Subsequently, 50 U of Mung BeenNuclease was added to the above solutions, followed by incubation at 37°C. for 30 minutes, for the purpose of degradation of the portions ofsingle-stranded DNA through the degradation by ExoIII. Then, theresultant solutions were subjected to extraction with phenol/chloroformand precipitation with ethanol. The DNA termini were repaired withKlenow Fragment (terminal smoothing of DNA) to make the DNA terminicompletely smooth. Then, the resultant solutions were subjected toextraction with phenol/chloroform and precipitation with ethanol,followed by digestion with XhoI. Through the above procedures,translational regions of Ref-1 with various length-deletion in itsC-terminal region, with which the N-terminus was the XhoI-digestedterminus and the C-terminus was the smooth terminus, were obtained.

[0113] Three fragments, that is, one of these various DNA fragments,isolated and purified pET14b XhoI/BamHI-digested fragment and BamHILinker, were ligated to each other in order to construct plasmidsexpressing recombinant Ref-1 deletion mutants in E. coli. The basesequences of BamHI Linker are shown in the following. Every frame of theLinker was designed to contain termination codon. The preparation methodfor the Linker is the same as the above method by which various kinds ofdouble-stranded DNA with ligation sequence for each transcriptionalfactor were prepared in the above. BamHI Linker :5′-TAACTAACTAG-3′ (SEQID NO: 7) :3′-ATTGATTGATCCTAG-5′ (SEQ ID NO: 8)

[0114] Names for plasmids expressing recombinant Ref-1 deletion mutantsin E. coli were taken from the number of deleted amino acid residuesfrom their C-termini after the translation. The names are described inthe following in order of the number: pET/RefdC76 (76 amino acidresidues from the C-terminus were deleted), pET/RefdC91 (91 amino acidresidues from the C-terminus were deleted), pET/RefdC163 (163 amino acidresidues from the C-terminus were deleted) and pET/RefdC182 (182 aminoacid residues from the C-terminus were deleted).

[0115] (c) Preparation of pET/RefdC230, pET/RefdC247 and pET/RefdC278

[0116] Through the same method as described in the above, pET/RefdC230(230 amino acid residues from the C-terminus were deleted), pET/RefdC247(247 amino acid residues from the C-terminus were deleted) andpET/RefdC278 (278 amino acid residues from the C-terminus were deleted)were constructed.

[0117] (d) Preparation of pET/RefdN41, pET/RefdN81, pET/RefdN121 andpET/RefdN161

[0118] A series of these recombinant Ref-1 N-terminal deletion mutantsexpressing in E. coli were amplified by Long-PCR method usingsynthesized oligonucleotides. Each base sequence of the synthesizedoligonucleotides is shown in the following. Each oligonucleotidepossesses individual digestive region by the restriction enzyme fromwhich each oligonucleotide takes its name. 5′RefdN41 XhoI primer:5′-ATGCCTCGAGATGCCAGCCCTGTATGAGGACC-3′ (SEQ ID NO: 9) 5′RefdN81 XhoIprimer: 5′-ATGCCTCGAGATGGATTGGGTAAAGGAAGAAGCC-3′ (SEQ ID NO: 10)5′RefdN121 XhoI primer: 5′-ATGCCTCGAGATGCCTTCGGACAAGGAAGGGT-3′ (SEQ IDNO: 11) 5′RefdN161 XhoI primer: 5′-ATGCCTCGAGATGTTTGACTCGTTTGTGCTGGTA-3′(SEQ ID NO: 12)

[0119] The synthesized oligonucleotides were heated at 55° C. for 8hours to be deprotected. The deprotected oligonucleotides weresubdivided into aliquots, dried under vacuum and dissolved in diluted (1in 10) buffer of TE (10 mM Tris-HCl (pH 7.9), 1 mM EDTA).

[0120] One of the above four kinds of oligonucleotides and 3′Ref-1 BamHIprimer were combined and subsequent procedures were the same aspreviously described to conduct PCR. The amplified DNA fragments wereindividually digested by Xhoi and BamHI (TOYOBO) and directly subjectedto agarose gel electrophoresis. A part of the agarose gel containing theDNA fragments was isolated, followed by phenol/chloroform extraction andethanol precipitation to purify the DNA fragments. One of these variousDNA fragments, isolated and purified pET14b XhoI/BaHI-digested fragmentand BamHI Linker, were ligated to each other in order tb constructpET/RefdN41 (41 amino acid residues from the N-terminus were deleted),pET/RefdN81 (81 amino acid residues from the N-terminus were deleted),pET/RefdN121 (121 amino acid residues from the N-terminus were deleted)and pET/RefdN161 (161 amino acid residues from the N-terminus weredeleted), respectively.

[0121] (e) Preparation of pET/RefdN41dC163, pET/RefdN41dC182 andpET/RefdN41dC213

[0122] First, pET/RefdN41 was digested with PvuII and XhoI, andsubjected to agarose gel electrophoresis. A part of the agarose gelcontaining only translational region was isolated. On the other hand,pET/RefdC163, pET/RefdC182 and pET/RefdC213 were digested with PvuII andBamHI, and subjected to agarose gel electrophoresis. Each part of theagarose gel containing only translational region was individuallyisolated and prepared. The same procedures, as described in the above,were conducted to ligate to each other in order to constructpET/RefdN41dC163 (41 amino acid residues from the N-terminus weredeleted and 163 amino acid residues from the C-terminus were deleted),pET/RefdN841dC182 (41 amino acid residues from the N-terminus weredeleted and 182 amino acid residues from the C-terminus were deleted)and pET/RefdN41dC213 (41 amino acid residues from the N-terminus weredeleted and 213 amino acid residues from the C-terminus were deleted),respectively.

[0123] (f) Preparation of pET/ RefdN51dC182, pET/RefdN61dC182,pET/RefdN71dC182, pET/RefdN81dC163 and pET/RefdN81dC182

[0124] The same procedures, as described in the above, were conducted toconstruct each deletion mutant.

EXAMPLE 13 Binding Assay of Mutational Recombinant Proteins of Ref-1Expressed in E. coli Against E3330

[0125] First, wild-type recombinant proteins of Ref-1 and mutationalrecombinant proteins of Ref-1 were expressed in E. coli and purifiedusing His Bind Resin or Glutathione Sepharose 4B. Then, each purifiedprotein was subjected to SDS-PAGE and stained using Rapid Stain CBB. Theobtained results are shown in FIGS. 8 and 9. A series of C-terminaldeletion mutants are shown in FIG. 8. A series of N-terminal deletionmutants and a series of both-sided C-terminal and N-terminal deletionmutants are shown in FIG. 9. In the FIG. 8, Lanes from Lane 1 to Lane 9were wild-type (WT), dC50, dC76, dC91, dC157, dC163, dC182, dC213 andGST-dC213 in this order, when electrophoresis was conducted. Theirmolecular weights were about 40 kDa, 37 kDa, 36 kDa, 35 kDa, 28 kDa, 26kDa, 23 kDa, 19 kDa and 42 kDa, respectively. In the FIG. 9, Lanes fromLane 1 to Lane 9 were wild-type (WT), dN41, dN81, dN 121, dN 161,GST-dN81dc182, GSTdN41dC213, GST-dN81dC213 and GST in this order, whenelectrophoresis was conducted. Their molecular weights are about 40 kDa,36 kDa, 32 kDa, 28 kDa, 22 kDa, 36 kDa (a band appearing just left sideof Lane 7), 37 kDa, 33 kDa and 28 kDa, respectively.

EXAMPLE 14 Identification of E3330-binding Domain Using MutationalRecombinant Proteins of Ref-1

[0126] Which kinds of Ref-1 deletion mutant proteins were bound to E3330was investigated to identify E3330-binding domain. Therefore, bindingassays were performed using E3330-immobilized SG particles. Theexperimental scheme was the same as illustrated in the FIG. 2.Concerning the elution procedure, two kinds of processes were conductedin this experiment. One is that 2 μg of each purified Ref-1 deletionmutant protein was mixed with E3330-immobilized SG particles, followedby standing still for 30 minutes in an ice-water bath; the proteinbinding to the particles was eluted with an elution buffer containing 1M KCl; and then, each eluate was subjected to 12.5% SDS-PAGE andexistence of proteins was detected by silver staining. The other one isthat 2 μg of each purified Ref-1 deletion mutant protein was mixed withE3330-immobilized SG particles, followed by standing still for 30minutes in an ice-water bath; 1×SDS sample dye was added to theparticles to which each kind of proteins was bound; and the resultantsuspension was directly boiled so that the protein binding to theparticles was eluted; and then, each eluate was subjected to 12.5%SDS-PAGE and existence of proteins was detected by CBB staining. As aresult, it was suggested that the following recombinant deletion mutantproteins of Ref-1 were bound to E3330-immobilized SG particles. In aprocess using an elution buffer containing 1 M KCl, the binding proteinswere wild-type (about 40 kDa), dC76 (about 36 kDa), dC91 (about 35 kDa),dC163 (about 26 kDa), dC182 (about 23 kDa), dC213 (about 19 kDa) andGST-dC213 (about 42 kDa). In the other process using a direct boilingmethod, the binding proteins were wild-type (about 40 kDa), dN41 (about36 kDa), dN81 (about 32 kDa), GST-dN81dC182 (about 36 kDa) andGST-dN81dC213 (about 33 kDa). These obtained results are shown in FIGS.10 and 11.

[0127] From the above results, Ref-1, that is a protein consisting of318 amino acid residues in its whole length, does not bind to E3330 incase where 231 or more amino acid residues from its C-terminus aredeleted or in case where 72 or more amino acid residues from itsN-terminus are deleted. Therefore, it was revealed that the amino acidsequence of at least 72 a.a. to 88 a.a. participated in the bindingactivity of Ref-1 against E3330, as shown in FIG. 12. Actually,recombinant protein possessing only the concerned amino acid sequence(72 a.a. to 88 a.a.) was synthesized and investigated on its bindingproperties using E3330-immobilized particles, resulting in confirmationof its binding ability against E3330. The amino acid sequence of 72 a.a.to 88 a.a. is as follows SEQ ID NO:13; length of sequence: 17 aminoacids): LRAWIKKKGLDWVKEEA.

[0128] These results indicated that the intracellular receptor to E3330was isolated and purified using E3330-immobilized particles and it isclear that the present invention is extremely useful for isolation andpurification of proteins.

EXAMPLE 15 Immobilization of DM852 to SG-EGDE Particles

[0129] Five mg of SG-EGDE particles, were prepared as described above,was washed three times with 1 ml of DH₂O. After washing, 500 μl of DH₂Osolution containing 5 μmol of DM852 was added to the packed SG-EDGE,followed by reaction at 37° C. for 24 hours, in order to immobilizeDM852 to epoxy groups of EGDE on the surfaces of SG-EGDE particles.After the reaction was finished, the particles were washed three timeswith 500 μl of H₂). The drug-immobilized particles were stored at 4° C.in a dark place with 500 μl of H₂O. The centrifugation procedure forwashing was conducted at 12,000×g for 3 minutes at room temperature.Under these reaction conditions about 0.1 mmol of DM852 was immobilizedonto 1 g of the SG-EGDE particles. The above immobilized amount of DM852was obtained by subtracting the amount of DM852 not bound to the SG-EGDEparticles frown the starting amount of DM852. DM852 shows the maximumabsorption at the wave-length of 265.0 nm, so that each amount of DM852can be determined by measuring an absorbance at the wave-length of 265.0nm on each sample; such as the starting solution, not binding fractionand washing fractions. The measurement on the absorbance was conductedwith DU-64 Spectrophotometer (BECKMAN).

Preparation of a Crude Nuclear Extract, Cytoplasmic and MembraneFractions

[0130] The culture of LP101 stroma cells, (5×10 ⁸ cells), which werecultured in a 150 mm dishes, was scraped and the collected cells wasconducted using 15-ml-centrifugation tubes and washed two times withPBS(−) at 300×g; for 5 minutes at 4° C. Then, the final packed cellvolume (PCV) was measured. Buffer A (10 mM Tris-HCl pH 7.4, 1 mM MgCl₂ 1mM EDTA, 0.5 mM DTT, 1 mM PMSF, 1 μgl/ml pepstain A, 1 μgl/mlleupeptin), four times larger volume of the PCV, was added to the packedcells to suspend the cells. The cell suspension was allowed to standstill on ice for 10 minutes so that the cells were swollen. The cellmembranes of the swollen cells were broken by 20 strokes using a15-ml-B-type Dounce homogenizer (IWAKI), transferred to15-ml-centrifugation tubes (IWAKI) and add NaCl (final concentration0.15M). Then, it was centrifuged at 840×g for 10 minutes at 4° C. forthe purpose of separating a nuclear fraction (pellet) from a membraneand cytoplasmic fraction (supernatant).

[0131] Buffer A, five times more volume of the PCV, was added to theisolated nuclear fraction to re-suspend the nuclei. The nuclearsuspension was centrifuged at 4,000×g for 5 minutes at 4° C. for thepurpose of removing the contaminated cytoplasmic fraction. The obtainednuclear pellet was dispersed In Buffer C (20 mM Tris-HCl pH 7.4, 420mMNaCI₂ 1 mM MgGl₂, 0.2 mM EDTA, 10% (v/v)glycerol, 0.5 mM DTT, 1 mM PMSF,1 μg/ml pepstatin A, 1 μg/ml leupeptin), the same volume as the PCV, andthoroughly suspended by 10 strokes using a B-type Dounce homogenizer.The suspension was slowly stirred for 30 minutes at 4° C. for thepurpose of extracting nuclear components. The extract was transferred toa 15-ml-centrifugation tube and centrifuged at 12,000×g for 30 minutesat 4° C. The obtained supernatant was dialyzed two times against 500 mlof Buffer E (10 mM Tris-HCl pH 7.4, 100 mM NaCl₂ 1 mM MgCl₂, 1 mM CaCI₂,0.2 mM EDTA, 10% (v/v)) glycerol, 0.1% NP-40, 0.5 mM DTT, 1 mM PMSF, 1μg/ml pepstatin A, 1 μgl/ml leupeptm) for 6 hours at 4° C.

[0132] On the other hand, the cytoplasmic fraction was, transferred toan ultra-centrifugation tube (BECKMAN NO: 344057) and ultra-centrifugedat 100,000×g for one hour at 4° C. (BECKMAN: Rotor Type SW50.1) for thepurpose of separating a membrane (pellet) from a cytoplasmic fraction(supenatant).

[0133] The obtained cytoplasmic fraction was dialyzed in the same manneras the above procedure for the nuclear extract.

[0134] Buffer A, two times more volume of the PCV, was added tore-suspend the obtained membrane fraction and Octylglucoside (finalconcentration 3%) was added for the purpose of solubilizing the membranefraction. The suspension was slowly stirred for four hours at 4° C., andultra-centrifuged at 100,000×g for one hour at 4° C. (BECKMAN: RotorType SW50.1). Then, the solubilized membrane fraction was dialyzed inthe same manner as the above procedure for the nuclear extract.

[0135] After the completion of the dialyses, the nuclear extract, thecytoplasmic fraction and the membrane fraction even subdivided intoappropriate aliquots and stored at −80° C. until the use of them.

[0136] In usual preparation, about 5 ml (5 mg/ml) of the nuclear extract15 ml (3 mg/ml) of the cytoplasmic fraction and 15 ml (2 mg/ml) of themembrane fraction, respectively were obtained in the scale of thisworking example.

Isolation of Proteins Using Microspheres

[0137] Microspheres and the extract and each fraction were mixed andcentrifuged to separate substances binding to DM852 which wasimmobilized on particles from the mixture. The centrifiugation procedurefor separation was conducted at 12,000×g for 3 minutes at 4° C. Allprocedures in the above were conducted at 4° C.

[0138] First, 1 mg of each DM852-not-immobilized SG-EGDE particles andDM-852-immobilized SG-EGDE particles mixture, which include 0.03 mg ofDM852-immobilized SG EGDE particles, were washed three times with 400 μlof Buffer E. These particles were used as a reference control againstDM852 immobilized SG EGDE particles. To these washed particles DM852-notimmobilized SG EGDE and DM852-immobilized SG-EGDE particles mixture, 500μl each of the extract and fractions, were added and mixed. Thesemixtures were allowed to be standing still for 4 hours withintermittently rotating in order to bind proteins possessingDM852-binding abilities to DM852 which was immobilized on the particles.The mixture was centrifuged and the supernatant was discarded. Thepellet was washed three times with 500 μl of Buffer E to removenon-specific binding substances as much as possible. Subsequently thewashed pellet was eluted with 50 μl of Buffer E+ which contained 10 mMDM852 in Buffer E, so that the proteins possessing DM852-bindingabilities were dissociated and eluted from DM852 immobilized on theparticles. The washed solution and eluted solution were stored at −80°C.

[0139] The detection of the proteins possessing DM852-biinding abilitieswas conducted by electrophoresis on a 10% SDS-polyacrylamide gel(SDS-PAGE) using 5 μl obtained from the DM852-immobilized SG-EGDEparticles mixture and DM852-not-immobilized SG-EGDE particles used as areference control. In this experiment, 4×SDS special dye (200 mMTns-HCl(pH 6.8), 500 mM β-mercaptoethanol (β-ME), 8% SOS, 0.4% BPB) wasused instead of 4×SDS sample dye in order to prevent the electrochoresisfrom being disordered due to such high concentration of the salts. Theelectrophoresed gel was subjected to silver staining and the proteinsspecifically binding to DM852 were identified in comparison with theresults of the reference control. As a result, in the membrane fraction,two protein bands with molecular weights of about 70 kDa aloud 80 kDawhich were not observed in the reference control were clearly observed,suggesting that the proteins were specifically binding to DM852. TheDM852-binding proteins were obtained about 10 ng each in the aboveprocedure. In the nuclear extract and cytoplasmic fractions any specificbands were not detected.

Evaluation of Specific-binding Abilities Against DM852

[0140] To confirm that the protein was specifically bound to DM852 acompetitive binding-inhibitory experiment was conducted.

[0141] In the step of the above procedure for the addition of themembrane fraction to SG-EGDF particles, free DM852 at 100 times and 400times more moles of the DM852 immobilized on the particles were addedsimultaneously. Then, the protein possessing specifically bindingabilities to DM852 immobilized on the particles would be bound to freeDM852, resulting in a lower yield in the isolation using the particles.As a result, it was confirmed that the yield of the protein with amolecular weight of about 70 kDa and 80 kDa were lowered, indicatingthat the proteins were specifically bound to DM852.

EXAMPLE 16 Immobilization of H-9 With a Spacer

[0142] The spacer-immobilized particles, that are SG-EGDE, particles,were prepared as described above. Five mg of SG-EGDE particles werewashed with 200 μl of H₂O three times and then 500 μl of H₂O containingH-9 (final conc. 30 mM) was added to the particles in order toimmobilize H-9 to epoxy group of EGDE on the surface of SG-EGDEparticles. After 12 hours incubation at 37° C. in a dark room, theparticles were washed three times with 400 μl of H₂O through acentrifugation manner. Then, the particles were dispersed in 0.5 MTris-HCL buffer (pH 8.5), allowed to be standing still 4° C. for atleast 24 hours and used in order to thoroughly mask the intact epoxygroups, on the surface of SG-EGDE particles. The drug-immobilizedparticles were stored at 4° C. in a dark place. The centrifugationprocedure for washing was conducted at 15,000×g for 5 minutes at roomtemperature. Under these reaction conditions about 0.14 mmol of H-9 wasimmobilized onto 1 g of the SG-EGDE) particles.

Isolation of Proteins Using Microspheres

[0143] HeLa cell nuclear extracts (NE) were prepared according to themethod as described previously (Dignam, J. D., Lebovitz, R. M. andRoeder, R. G. (1983). Accurate transcription by RNA pol II in a solubleextracts from isolated mammalian nucleic. Nucleic Acids Res 11.1475-1489). Three hundred micrometer of NE was incubated for 30 minutesat 4° C. with 1.0 mg of H-9-not-immobilized SG-EGDE particles andH-9-immobilized SG-EGDE particles which have been washed three timeswith 200 μl of HGKEDN (20 mM HEPES (pH 7.9), 10% (V/V) glycerol, 0.1MKCL, 0.2 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40(NP-40)). TheH9-not-immobilized SG.EGDE particles were dispersed in 1 ml of 1 M TrisHCl buffer solution (pH 7.4) and allowed to be standing still at 4° C.for at least 24 hours in order to mask epoxy groups of EGDE. Theseparticles were used as a reference control against H-9-immobilized SGEGDE particles. The mixture was centrifuged and the supernatant wasdiscarded. The pellet was washed three times with 500 μl of HGKEDN toremove non-specific binding substances as much as possible.

[0144] Subsequently, the washed pellet was eluted three times withHGKEDN containing 1 M KCl or 3 mM H-9 or 3 mM H-9, so that the proteinspossessing H-9-binding abilities were dissociated and eluted from H-9 isimmobilized on the particles. The wash solution and eluate solution werestored at −80° C.

[0145] The detection of the proteins possessing H9-binding abilities wasconducted by electrophoresis on a 10% SDS-polyacrylamide gel (SDS-PAGE)using 20 μl each of the first, second or third eluate solution obtainedfrom the H-9-immobilized SG-EGDE particles and H9-not-immobilized SGEGDE particles used as a reference control. In this experiment, 4×SDSspecial dye (200 mM Tns-HCl pH 6.8), 500 mM b-mercaptoethanol (b-ME), 8%SDS, 0.4% BPB) was need instead of 4×SDS special dye in order to preventthe electrophoresis from being disordered due to such high concentrationof the salts. The electrophoresed gel was subjected to silver stainingand the protein specifically binding to H-9 were identified incomparison with the results of the reference control. As a result, aprotein band with a molecular weight of about 30 kDa which was notobserved in the reference control was clearly observed, suggesting thatthe protein was specifically binding to H-9. The same protein band wasobtained by elution using H-8 or H-9, suggesting that the 30 kDa proteinwas bound to the H-9-immobilized SG-EGDE particles in a specific manner.

Evaluation of Specific Binding Abilities Against H-9

[0146] A competition experiment was conducted to confirm that theprotein with a molecular weight of about 30 kDa in HeLa cell nuclearextract was specifically bound to H-9. In the step of a procedure forthe addition of HeLa cell nuclear extracts to SG-EGDE particles, whenfree H-9 or H-8 were added simultaneously to H-9-immobilized SG-EGDEparticles with indicated concentrations, the addition of the drugsresulted in a lower yield of the 30 kDa protein in the isolation usingthe particles. Thus, it was confirmed that the yield of the protein witha molecular weight of About 30 kDa was lowered, indicating that theprotein was specifically bound to H-9 or H.8.

EXAMPLE 17 Immobilization of an Amino Derivative of DQ2511 (Ecabapide)With a Spacer

[0147] (a) (Induction of an amino derivative of DQ2511)

[0148] As DQ2511 does not possess an appropriate functional group, it isdifficult to immobilize DQ2511 onto SG-EGDE particles. Therefore,NH₂-DQ2511 was synthesized through the induction of an amino group intoDQ2511.

[0149] (b) (Immobilization of NH₂-DQ2511 to SG-EGDE particles)

[0150] Ten mg of SG-EGDE particles described above was washed threetimes with 500 μl of H₂O through a centrifugation procedure. Afterwashing, 500 μl of H₂O solution containing 4 μmol of NH₂-DQ2511 wasadded to the packed SG-EGDE particles to disperse the SG-EGDE particlesin the above solution, followed by reaction at 37° C. for 24 hours, inorder to immobilize NH₂-DQ2511 to epoxy groups of EGDE on the surfacesof SG-EGDE particles. After the reaction was finished, the particleswere washed three times with 400 μl of H₂O through a centrifugationprocedure. Then, the particles were dispersed in 500 μl of 0.5 MTris-HCl buffer solution (pH 8.5), allowed to be standing still at 4.Cfor at least 24 hours and used, in order to thoroughly mask the intactepoxy groups on the surfaces of SG-EGDE particles. The drug-immobilizedparticles were stored at 4° C. in a dark place. The centrifugationprocedure for washing was conducted at 15,000×g for 5 minutes at roomtemperature. Under these reaction conditions about 0.06 mmol ofNH₂-DQ2511 was immobilized onto 1 g of the SG-EGDE particles. The aboveimmobilized amount of NH₂-DQ2511 was obtained by subtracting the amountof NH₂-DQ2511 not bound to the SG-EGDE particles from the startingamount of NH₂-DQ2511 NH2-DQ2511 shows the maximum absorption at thewave-length of 275.0 nm, so that each amount of NH₂-DQ2511 can bedetermined by measuring an absorbance at the wave-length of 275.0 nm oneach sample, such as the starting solution, not-binding fraction andwashing fractions. The measurement on the absorbance was conducted withDU-64 Spectrophotometer (BECKMAN).

Preparation of a Crude Cytoplasmic Fraction From HeLa Cell

[0151] The culture medium suspension of HeLa cells (3×10⁹ cells), whichwere cultured in a suspension scale of 8 liters, was centrifuged using500-ml-centrifugation tubes (NARGEN) at 500×g for 10 minutes at 4° C.for the purpose of collecting the cells. The collected cells were washedtwo times with PBS(−). The washing procedure was conducted using50-ml-centrifugation tubes and the centrifugation conditions were At700×g for 5 minutes at 4° C. Then, the final packed cell volume (PCV)was measured. Buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5mM DTT), four times larger volume of the PCV, was added to the packedcells to suspend the cells. The cell suspension was allowed to standstill on ice for 20 minutes 90 that the cells were swollen. The cellmembranes of the swollen cells were broken by 20 strokes using a40-ml-B-type Dounce homogenizer (WHEATON), transferred to a50-ml--centrifugation tube (NARGEN) and centrifuged at 4,200×g for 6minutes at 4° C. for the purpose of separating a nuclear fraction(pellet) and a cytoplasmic fraction (supernatant).

[0152] The cytoplasmic fraction was transferred to a50-ml-centrifugation tube and centrifuged at 20,000×g for 1 hour at 4°C. The obtained supernatant was dialyzed three times against 500 ml ofBuffer 0.05 HKMGED (20 mM Hepes pH 7.9, 10% (V/V) glycerol, 0.05 M KCl,1 mM MgCl₂, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT) for 2 hours at 4° C.After the completion of the dialyses, the cytoplasmic fraction of HeLacell was centrifuged at 20,000×g for 1 hour at 4° C. The obtainedsupernatants was used as the sample of cytoplasmic fraction of HeLacell. This sample was subdivided into appropriate aliquots and stored at−80° C. until the use of them. In usual preparation, about 20 ml of thecytoplasmic fraction at the protein concentration of 10 mg/ml, wasobtained in the scale of this working example,

Preparation of a Crude Lysate From Rat Dorsal Root Ganglia (DRG) Cell

[0153] Dorsal root ganglia cells isolated from rats were washed twotines with PBS(−). The washing procedure was conducted using15-ml-centrifugation tubes and the centrifugation conditions were at700×g for 5 minutes at 4° C. Then, the final packed cell volume (PCV)was measured, PBS(−), four times larger volume of the PCV, was added tothe packed cells to suspend the cells. The cells were broken completelyby homogenize, transferred to 1.5 ml tubes (eppendorf) and centrifugedat 136,000×g for 1 hour at 4° C. In usual preparation, the supernatantof lysate including 1 mg/ml of protein was obtained.

[0154] To concentrate the crude lysate of DRG cell, the proteinscontained in it were salted out with saturated ammonium sulfate. Aftercentrifugation at 20,000×g for 1 hour at 4° C., the supernatant wasremoved. The pellet was dissolved with 0.05 HKMGED (20 mM Hepes pH 7.9,10% (V/V) glycerol, 0.05 M KCl, 1 mM MgCl₂, 0.2 mM EDTA, 0.4 mM PMSF, 1mM DTT) to be one fourth volume of starting lysate. The concentratedlysate was dialyzed three times against 500 ml of Buffer 0.05 HKMGED (20mM Hepes pH 7.9, 10% v/v) glycerol, 0.05 M ACT, 1 me MgClz, 0.2 mM EDTA,0.4 MU PMSF, 1 EM DTT) for 2 hours at 4° C.

[0155] After the completion of the dialyses, the concentrated lysate ofDRG cell was centrifuged at 20,000×g for 1 hour at 4° C. The obtainedsupernatant was used as the sample of lysate of DRG cell. This samplewas subdivided into appropriate aliquotd and stored at −80° C. until theuse of them. In usual preparation, the concentrated lysate including 3mg/ml of protein was obtained

Isolation of Proteins From a Cytoplasmic Fraction of HeLa Cell UsingMicrospheres

[0156] A process of isolation and purification of DQ2511-bindingproteins using DQ2511-immobilized particles is illustrated above. Beforeusing, cytoplasmic fraction of HeLa cell was diluted to ten third volumewith Buffer 0.05 HKMGEDN (20 mM Hepes pH 7.9, 10% v/v glycerol, 0.05 MKCl, 1 mM MgCl:, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT, 0.01% NP-40).

[0157] (a) microspheres and the diluted cytoplasmic fraction were mixedand centrifuged to separate substances binding to DQ2511 which wasimmobilized on particles from the mixture. The centrifugation procedurefor separation was conducted at 15,000×g for 5 minutes at 44° C. Allprocedures in the above were conducted at 4° C.

[0158] (b) First, 1 mg each of DQ2511-not-immobilized SG-EGDE particlesand DQ2511-immobilized SG-EGDB particles were washed three times with400 μl of Buffer 0.05 HKMGEDN (20 mM Hepes pH 7.9, 10% (V/V) glycerol,0.05 M KCl, 1 mM MgCl₂, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT, 0.01%NP-40). The DQ2511-not-immobilized SG-EGDE particles were dispersed in 1ml of 0.5 M Tris-HCl buffer solution (pH 8.5) and allowed to be standingstill at 4° C. for at least 24 hours in order to mask epoxy groups ofEGDE. These particles were used as a reference control against DQ2511-immoblllzed SG-EGDE particles. To these washed DQ2511-not-immobilizedSG-EGDE particles and DQ2511-immobilized-SG-EGDE particles, 1 ml of thediluted cytoplasmic fraction was added and mixed. These mixtures wererotated for 1 hour at 4° C. in order to bind proteins possessingDQ2511-binding abilities to DQ2511 which was immobilized on theparticles. The mixture was centrifuged and the supernatant wasdiscarded. The pellet was washed seven times with 200 μl 0.05 HKMGEDN toremove non-specific binding substances as much as possible.Subsequently, the washed pellet was eluted two times with 20 μl ofBuffer 1.0 HKMGEDN (20 mM Hepes pH 7.9, 10% (v/v) glycerol, 1.0 M KCl, 1mM MgCl₂, 0.2 mM EDTA, 0.4 mM PMSF, 1 mM DTT, 0.01% NP-40), so that theproteins possessing DQ2511-hinding abilities were dissociated and elutedfrom DQ2511 immobilized on the particles. The wash solution and eluatesolutions were stored at −80° C.

[0159] (c) The detection of the proteins possessing DQ2511-bindingabilities were conducted by electrophoresis on a 8% SDS-polyacrylamidegel (SDS-PAGE) using 5 μl each of the first, second or third eluatesolution obtained from the DQ2511-immobilized SG-EGDE particles andDQ2511-not-immobilized SG-EGDE particles used as a reference control.The electrophoresed gel was subjected to silver staining and theproteins specifically binding to DQ2511 were identified in comparisonwith the results of the reference control. As a result, in thecytoplasmic fraction a protein band with a molecular weight of about 110kDa which was not observed in the reference control was clearlyobserved, suggesting that the protein was specifically binding toDQ2511.

[0160] In the above procedure, about 20 ng of 110 kDa DQ2511-bindingprotein was obtained from 1 mg of the DQ2511-immobilized SG-EGDEparticles.

Isolation of Proteins From a Lysate of Rat DRG Cell Using Microsphere

[0161] Microspheres and the concentrated crude lysate of rat DRG cellwere mixed and centrifuged to separate substances binding to DQ2511which was immobilized on particles from the mixture. The procedures ofbinding, washing and detection of protein were performed as describedabove. As a result, a protein with a molecular weight about 170 kDa wasobtained as the protein possessing DQ2511-binding ability. In the aboveprocedure, about 10 ng of 170 kDa DQ2511-binding protein was obtainedfrom 1 mg of the DQ2511-immobilized SG-EGDE particles.

Evaluation of Specific-binding Abilities Against DQ2511

[0162] The following competitive binding-inhibitory experiment wasconducted to confirm that the 110 kDa protein in the cytoplasmicfraction of HeLa cell and 170 kDa protein in the lysate of rat DRG cellbound to DQ2511 specifically.

[0163] When in the step of a procedure for the addition of either thecytoplasmic fraction or crude lysate of rat DRG cell to SG-EGDEparticles, free NH₂-DQ2511 at 50 times, 150 times or 500 times moremoles than the immobilized NH₂-DO2511 were added simultaneously. Theprotein possessing specifically binding abilities to DQ2511 immobilizedon the particles would be bound to free NH₂-DQ2511, resulting in a loweryield in the isolation using the particles. As a result, thepurifications of the protein with a molecular weight of about 110 kDaand the protein with a molecular weight of about 170 kDa from theDQ2511-immobilized particles were inhibited with dose dependence of freeNH₂-DQ2511, indicating that the proteins were specifically bound toNH₂-DQ2511.

EXAMPLE 18 Immobilization of KF43389 to SG-EGDE Particles

[0164] Five mg of SG-EGDE particles was washed three times with 1 ml of1.4-dioxane through a centrifugation procedure. After washing, 500 μl of1.4-dioxane solution containing 15 μmol of KF49389 was added to thepacked SG-EGDE particles to disperse the SG-EGDE particles in the abovesolution, followed by reaction at 37° C. for 48 hours, in order toimmobilize KF49389 to epoxy groups of EGDE on the surfface of SG-EGDEparticles. After the reaction was finished, the particles were washedthree times with 500 μl of 1.4-dioxane and three times with waterthrough a centrifugation procedure. The drug-immobilized particles werestored at 4° C. in a dark place with 500 μl of water. The centrifugationprocedure for washing was conducted at 15,000×g for 5 minutes at roomtemperature.under these reaction conditions about 0.15 mmol of KF49389was immobilized onto 1 g of the SG-EGDE particles. The above immobilizedamount of KF49389 was obtained by subtracting the amount of KF49389 notbound to the SG-EGDE particles from the starting amount of KF49389.KF49399 shows the maximum absorption at the wave-length of 282.0 nm sothat each amount of KF49389 can be determined by measuring an absorbanceat the wave-length of 282.0 nm on each sample, such as the startingsolution, not-binding fraction and washing fractions. The measurement onthe absorbance was conducted with DU-64 Spectrophotometer (BECKMAN).

Preparation of a Crude Nuclear Extract and a Cytoplasmic Fraction

[0165] The culture of MG63 cells (1×10⁹ cells), which were cultured in a150 mm dishes, was scraped and the collected cells was conducted using50-ml-centrifugation tubes and washed two times with PBS(−) at 3000×gfor 10 minutes at 4° C. Then, the final packed cell volume (PCV) wasmeasured. Buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mMDTT), two times larger volume of the PCV, was added to the packed cellsto suspend the cells. The cell suspension was allowed to stand still onice for 10 minutes so that the cells were swollen. The cell membranes ofthe swollen cells were broken by 20 strokes using a 40-ml-B-type Douncehomogenizer (WHEATON), transferred to a 50-mil-centrifugation tube(NARGEN) and centrifuged at 2000×g for 10 minutes at 4° C. for thepurpose of separating a nuclear fraction (pellet) from a cytoplasmicfraction (supernatant). Buffer A, five times larger volume of the PCV,was added to the isolated nuclear fraction to re-suspend the nuclei. Thenuclear suspension was centrifuged at 4,200×g for 6 minutes at 4° C. forthe purpose of removing the contaminated cytoplasmic fraction. Theobtained nuclear pellet was dispersed in Buffer C (20 mM Hepes pH 7.9,25% v/v glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM PMSF, 1mM DTT), the same volume as the PCV, and thoroughly suspended by 10strokes using a B-type Dounce homogenizer. The suspension was slowlystirred for 30 minutes at 4° C. for the purpose of extracting nuclearcomponents. The extract was transferred to a 50-ml-centrifugation tubeand centrifuged at 3,000×g for 10 minutes at 4° C. The obtainedsupernatant was dialyzed two times against one liter of Buffer D (20 mMHepes pH 7.9, 10% (V/V) glycerol, 0.05 M KCl, 0.2 mM EDTA, 1 mM MgCl₂,0.1 mM PMSF, 1 mM DTT) for 4 hours at 4° C.

[0166] On the other hand, the cytoplasmic fraction was transferred to anultra-centrifugation tube and ultra-centrifuged at 35 Krpm for one hourat 4° C. (BECKMAN: Rotor Type SW: 50.1). The obtained supernatant wasdialyzed in the same manner as the above procedure for the nuclearextract.

[0167] After the completion of the dialyses, the nuclear extract and thecytoplasmic fraction were subdivided into appropriate aliquots andstored at −80° C. until the use of them.

Isolation of Proteins Using Microspheres

[0168] (a) Microspheres and the cytoplasmic were mixed and centrifugedto separate substances binding to KF49389 which was immobilized onparticles from the mixture. The centrifugation procedure for separationwas conducted at 15,000×g for 5 minutes at 4° C. All procedures in theabove were conducted at 4° C.

[0169] (b) First, 0.5 mg each of KF49389-not-immobilized SG-EGDEparticles and KF49309-immobilized SG-EGDE particles were washed threetimes with 400 μl of Buffer D. These particles were used as a referencecontrol against KF49389-immobilized SG-EGDE particles. To these washedKF49389-not-immobilized SG-EGDE particles and KF49389-immobilizedSG-EGDE particles, 3 mg per 1 ml of the cytoplasmic fractionation, wasadded and mixed. These mixtures were allowed to be standing still for 4hours with rotating in order to bind proteins possessing KF49389-bindingabilities to KF49389 which was immobilized on the particles. The mixturewas centrifuged and the supernatant was discarded. The pellet was washedfour times with 500 μl of Buffer D to remove non-specific bindingsubstances as much as possible.

[0170] Subsequently, the washed pellet was eluted with 30 μl of Buffer E(20 mM Hepes pH 7.9, 10% (V/V) glycerol, 1 mM DTT, 0.2 mM EDTIA, 1 mMMgCl₂, 0.1 mM PMSF 1 mM DTT), so that the proteins possessingKF49389-binding abilities were dissociated and eluted from KF49389immobilized on the particles. The wash solution and eluate solution werestored at −80° C.

[0171] (c) The detection of the proteins possessing KF49389-bindingabilities was conducted by electrophoresis on a 10% SDS-polyacrylamidegel (SDS-PAGE) using 10 μl obtained from the KF49389-immobilized SG-EGDEparticles and KF49389-not-immobilized SG-EGDE particles used an areference control. In this experiment, 4×SDS special dye (200 mMTris-HCl (pH 6.8), 500 mM β-mercaptoethanol (β-ME), at SDS, 0.4% BPB)was used instead of 4×SDS sample dye in order to prevent theelectrophoresis from being disordered due to such high concentration ofthe salts. The electrophoresed gel was subjected to silver staining andthe proteins specifically binding to KF49389 were identified incomparison with the results of the reference control. As a result, inthe cytoplasmic fraction a protein band with a molecular weight of about55 kDa which was not observed in the reference control was clearlyobserved, suggesting that the protein was specifically binding toKF49389. This binding protein was obtained about 10 ng from each tubes.

Confirmation of the Binding Ability of KF49389-binding Protein

[0172] (a) The above procedure was repeated and finally 5 μg ofKF49389-binding protein was obtained. N-terminal amino acid sequence ofthe obtained protein was analyzed and three different sequences wasdetected. These amino acid sequences were analyzed using a computer anddetermined to be identical with the amino acid sequences of N-terminalregion of three different proteins. These proteins are conditionallynamed as protein A, B. C, respectively, These proteins were cloned usingPCR method from HeLa cells cDNA library, and subcloned into E. coliexpression plasmids pGEX4T-2 (Pharmacia) which would express the GST-Tagfused recombinant protein of A, B, C wild type. The expression plasmidswere introduced into E. coli BL21 (DE3) and protein expression wasinduced by addition of 0.4 mM IPTG. Purification of the GST-taggedproteins was performed according co the manufacturer's instructions(Pharmacia}.

[0173] (b) The binding ability of the recombinant protein obtained inthe above procedure against KF49389 was investigated using KF49389immobilized particles 400 ng per 500 μl of the recombinant protein, GSTalone or GST-Tagged protein A, B, C, was incubated with KF49389immobilized particles and performed as above, As a result, GST alone wasnot recovered from KF49389-immobilized particles, entirely. In contrast,recombinant proteins of GST-Tagged protein A, B, C were recovered fromKF49389-immobilized particles and especially GST-Tagged A was obtainedhigh efficiently against input recombinant protein (about 80%). Theseresults indicates that the protein A, B, C can bind with KF49389specifically and the protein A has a high affinity with KF49389.

EXAMPLE 19 Isolation of Proteins Using Microspheres

[0174] A process of isolation and purification of E3330-binding proteinsusing E3330-immobilized particles is illustrated in FIGS. 14 an 15.

[0175] (a) Microspheres and the crude nuclear extract or each fractionobtained through the fractionation use a phosphocellulose column in theworking example 5 were mixed and centrifuged to separate substancesbinding to E3330 which was immobilized on particles from the mixture.The centrifugation procedure for separation was conducted at 15,000×gfor 5 minutes at 4° C. All processes in the above were conducted at 4°C.

[0176] (b) First, 1 mg each of E3330-not-immobilized SG-EGDE particlesand E3330-immobilized SG-EGDE particles were washed three times until250 μl of Buffer D2 (20 mM HEPES (pH 7.9), 10% (V/V) glycerol, 0.125 MKCL 0 2 mM EDTA, 1 mM DTT) in Which KCl and glycerol concentrations were0.125 M and 10% instead of 0.1 M and 20%, respectively, in Buffer D. TheE3330-not-immobilized SG-EGDE particles were dispersed in 1 ml of 1 MTris-HCl buffer solution (pH 7.4) and allowed to be standing sell at 4°C. for at least 24 hours in order to mask epoxy groups of EGDE. Theseparticles were used as a reference control against E3330-immobilizedSG-EGDE particles To these washed E3330-not-immobilized SG-EGDEparticles and E3330-immobilized SG-EGDE particles, 100 μl each of eachof the nuclear extract, P.1, P.3 P.5, or P1.0 which were diluted withbuffer D2 to a concentration of 0.16 mg/ml was added and mixed Thesemixtures were allowed to be standing still for 30 minutes withintermittenly stirring at intervals of 10 minutes in order to bindproteins possessing E3330-binding abilities to E3330 which wasimmobilized on the particles. The mixture was centrifuged and thesupernatant was discarded. The pellet was washed five times With 250 μlof Buffer D2 to remove non-specific binding substances as much aspossible. Subsequently, the washed pellet was eluted with 20 μl ofBuffer D containing 1M KCl, so that the proteins possess E3330-bindingabilities were disassociated and eluted from E3330 immobilized on theparticles. The wash solution and eluate solution were stored at −80° C.

[0177] (c) The detection of the protein possessing E3330-bindingabilities was conducted by electrophoresis on a 10% SDS-polyacrylamidegel (SDS-PAGE) using 10 μl of the eluate solution obtained from theE3330-mobilized SG EGDE particles and E3330-not-immobilized SG-EGDEparticles used as a reference control. In this experiment, 4×SDS Specialdye (200 mM Tris-HCl (pH 6.8), 500 mM β-mercaptoethanol (β-ME), 8% SDS,0.4% BPB) was used instead of 4×SDS sample dye in order to prevent theelectrophoresis from being disordered due to such high concentration ofthe salts. The electrophoresed gel was subjected to silver staining andthe proteins specifically binding to E3330 were identified in comparisonwith the results of the reference control. As a result, from thepurification using nuclear extract, three protein bands with a molecularweight of about 60, 38, and 27 kDa which were not observed in thereference control were clearly observed, suggesting that the proteinsrevere specifically binding to E3330. From the purification using P.1,there was not observed any specific protein band specifically elutedfrom E3330-immobilized particles.

[0178] The above procedure was repeated and finally 5 μg ofE3330-binding protein with a molecular weight of about 38 kDa wasobtained. From the purification using P.3, a protein band with amolecular weight of about 60 kDa which were not observed in thereference control were clearly observed. From the purification usingP.5, a protein hand with a molecular weight of about 38 kDa which werenot observed in the reference control were clearly observed. From thepurification using P.1.0, a protein band with a molecular weight ofabout 27 kDa which were not observed in the reference control wereclearly observed.

EXAMPLE 20 Evaluation of Specific-binding Abilities Against E3330

[0179] Two kinds of experiments were conducted to confirm that theproteins with a molecular weight of about 60, 38, and 27 kDa in thenuclear extracts of Jurkat cells were specifically bound to E3330.

[0180] The first one was a competitive binding-inhibitory experiment.When in the step of a procedure for the addition of the nuclear extractsto SG-EGDE particles free NH₂-E3330 at the same moles of the NH₂-E3330immobilized on the particles or free NH₂-E3330 at ten times more molesthan the immobilized NH₂-E3330 were added simultaneously, the proteinpossessing specifically binding abilities to E3330 immobilized on theparticles would be bound to free NH₂-E3330, resulting in a lower yieldin the isolation using the particles. As a result, it was confirmed thatthe yield of the proteins with a molecular weight of about 60, 38, and27 kDa was lowered, indicating that the proteins were specifically boundto E3330.

[0181] Next, the other experiment was conducted by varying the amount ofNH₂-E3330 Mobilized on the SG-EGDE particles. In the present study themaximum amount of the immobilized E3330 derivatives is 0.4 μmol per 1 mgof SG-EGDE particles. Under these conditions, about 5 to 6 molecules ofE3330 derivatives are immobilized on the 1 mm² of the surface of theparticles. This experimental study was conducted in case where theamount of the immobilized E3330′ derivatives was 0.2 μmol or 0,4 μmolper 1 mg of SG-EGDE particles. However, in the present invention,amounts of compound immobilized on the particles are varied depending onthe properties of the immobilized compounds, conditions ofimmobilization and so on. The amounts are not defined and are generallyvaried between a few molecules and hundred molecules. As a result, itwas confirmed that the yield of the proteins with a molecular weight ofabout 60, 38, and 27 kDa increased as the immobilized amount increased.The identification of the specific proteins were conducted byelectrophoresis using SDS-PAGE.

[0182] The invention has been described in detail with reference topreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure, maymake modifications and improvements within the spirit and scope of theinvention as set forth in the following claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:13 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 1: Gly Leu Asp Trp Val Lys 1 5 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ala Ala Gly Glu Gly Pro Ala Leu TyrGlu Asp Pro Pro Asp 1 5 10 (2) INFORMATION FOR SEQ ID NO: 3: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gly Ala Val Ala Glu Asp Gly Asp Glu Leu 1 5 10 (2) INFORMATION FORSEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 319 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: Met Pro Lys Arg Gly Lys Lys Gly Ala Val AlaGlu Asp Gly Asp Glu 1 5 10 15 Leu Arg Thr Glu Pro Glu Ala Lys Lys SerLys Thr Ala Ala Lys Lys 20 25 30 Asn Asp Lys Glu Ala Ala Gly Glu Gly ProAla Leu Tyr Glu Asp Pro 35 40 45 Pro Asp Gln Lys Thr Ser Pro Ser Gly LysPro Ala Thr Leu Lys Ile 50 55 60 Cys Ser Trp Asn Val Asp Gly Leu Arg AlaTrp Ile Lys Lys Lys Gly 65 70 75 80 Leu Asp Trp Val Lys Glu Glu Asp AlaPro Asp Ile Leu Cys Leu Gln 85 90 95 Glu Thr Lys Cys Ser Glu Asn Lys LeuPro Ala Glu Leu Gln Glu Leu 100 105 110 Pro Gly Leu Ser His Gln Tyr TrpSer Ala Pro Ser Asp Lys Glu Gly 115 120 125 Tyr Ser Gly Val Gly Leu LeuSer Arg Gln Cys Pro Leu Lys Val Ser 130 135 140 Tyr Gly Ile Gly Asp GluGlu His Asp Gln Glu Gly Arg Val Ile Val 145 150 155 160 Ala Glu Phe AspSer Phe Val Leu Val Thr Ala Tyr Val Pro Asn Ala 165 170 175 Gly Arg GlyLeu Val Arg Leu Glu Tyr Arg Gln Arg Trp Asp Glu Ala 180 185 190 Phe ArgLys Phe Leu Lys Gly Leu Ala Ser Arg Lys Pro Leu Val Leu 195 200 205 CysGly Asp Leu Asn Val Ala His Glu Glu Ile Asp Leu Arg Asn Pro 210 215 220Lys Gly Asn Lys Lys Asn Ala Gly Phe Thr Pro Gln Glu Arg Gln Gly 225 230235 240 Phe Gly Glu Leu Leu Gln Ala Val Pro Leu Ala Asp Ser Phe Arg His245 250 255 Leu Tyr Pro Asn Thr Pro Tyr Ala Tyr Thr Phe Trp Thr Tyr MetMet 260 265 270 Asn Ala Arg Ser Lys Asn Val Gly Trp Arg Leu Asp Tyr PheLeu Leu 275 280 285 Ser His Ser Leu Leu Pro Ala Leu Cys Asp Ser Lys IleArg Ser Lys 290 295 300 Ala Leu Gly Ser Asp His Cys Pro Ile Thr Leu TyrLeu Ala Leu 305 310 315 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTCTCTCGAGATGCCGAAGC GTGGGAAAAA G 31 (2) INFORMATION FOR SEQ ID NO: 6: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:ATGCGGATCC TTACAGTGCT AGGTATAGGG T 31 (2) INFORMATION FOR SEQ ID NO: 7:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:TAACTAACTA G 11 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATTGATTGATCCTAG 15 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINALSOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGCCTCGAG ATGCCAGCCCTGTATGAGGA CC 32 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi)ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATGCCTCGAGATGGATTGGG TAAAGGAAGA AGCC 34 (2) INFORMATION FOR SEQ ID NO: 11: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATGCCTCGAG ATGCCTTCGG ACAAGGAAGG GT 32 (2) INFORMATION FOR SEQ IDNO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 12: ATGCCTCGAG ATGTTTGACT CGTTTGTGCT GGTA 34 (2)INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 13: Leu Arg Ala Trp Ile Lys Lys Lys GlyLeu Asp Trp Val Lys Glu Glu 1 5 10 15 Ala

What is claimed is:
 1. A microsphere which is prepared by coupling asubstance possessing physiological activity to a styrene-glycidylmethyacrylate polymer through a spacer, wherein at least one functionalgroup of any of the substance possessing physiological activity, thepolymer or spacer is converted to another type of functional group. 2.The microsphere according to claim 1, wherein the functional group is onthe particle.
 3. The microsphere according to claim 1, wherein thefunctional group is on the spacer.
 4. The microsphere according to claim1, wherein the functional group is on the substance possessingphysiological activity.
 5. The microsphere according to the claim 1,wherein the spacer is an ethylene glycol diglycidyl ether derivative. 6.The microsphere of claim 1, wherein the polymer consists of units ofstyrene and glycidyl methacrylate.
 7. The microsphere according to claim1, wherein the functional group is an epoxide.
 8. The microsphereaccording to claim 1, wherein the functional group is covalently boundto a nucleophile.
 9. The microsphere according to claim 1, wherein thefunctional group is converted to a hydroxy group, amino group, thiolgroup or carboxyl group.
 10. A microsphere comprising a substancepossessing physiological activity, a polymer and a spacer, wherein atleast one functional group of any of the substance possessingphysiological activity, the polymer or spacer is converted to anothertype of functional group.
 11. A process of preparing a microspherecomprising coupling a substance possessing physiological activity to astyrene-glycidyl methyacrylate polymer through a spacer, wherein atleast one functional group of any of the substance possessingphysiological activity, the polymer or spacer, is converted to anothertype of functional group.
 12. The process according to claim 11, whereinthe functional group is on the particle.
 13. The process according toclaim 11, wherein the functional group is on the spacer.
 14. The processaccording to claim 11, wherein the functional group is on the substancepossessing physiological activity.
 15. The process according to claim11, wherein the functional group is an epoxide.
 16. The processaccording to claim 11, wherein the functional group is covalently boundto a nucleophile.
 17. The process according to claim 11, wherein thefunctional group is converted to a hydroxy group, amino group, thiolgroup or carboxyl group.
 18. A process of isolating a substance that canadhere to a substance possessing physiological activity from a mixturecontaining the substance, comprising contacting the mixture with amicrosphere prepared by coupling the substance possessing physiologicalactivity to a polymer through a spacer, and isolating the substance fromthe mixture, wherein the substance is selected from the group consistingof DM852, H-9, DQ2511 and KF49389 and derivatives thereof.